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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.

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&rsquo, 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.

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.

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.

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/beta-catenin pathway. Mechanistically, TRAP1 maintenance of stemness is mediated by the regulation of Wnt/beta-catenin signaling, through the modulation of the expression of frizzled receptor ligands and the control of beta-catenin ubiquitination/phosphorylation. Remarkably, TRAP1 is associated with higher expression of beta-catenin and several Wnt/beta-catenin target genes in human CRCs, thus supporting the relevance of TRAP1 regulation of beta-catenin in human pathology. This study is the first demonstration that TRAP1 regulates stemness and Wnt/beta-catenin pathway in CRC and provides novel landmarks in cancer biology and therapeutics.

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NTRODUCTION: 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.

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. Cancer 2012;000:000–000. V C 2012 American Cancer Society.

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34+ cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34+ cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34+ leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34+ subfraction exhibiting the phenotype (CD34+/CD38−/CD123+/CD33+/CD90−) of leukemic stem cells. When transplanted into immunocompromised mice, CD34+ cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34+ fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34− cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Each year, nearly one million people develop colorectal cancer, and 50 % of them are expected to die because of cancer within 5 years of diagnosis. Over the last two decades, significant advances in screening, surgery, adjuvant chemotherapy, and patient monitoring have improved the 5-year survival rate. Colorectal cancer is a molecularly heterogeneous disease, and the analysis of its molecular signatures and the identification of colorectal cancer biomarkers will lead to better screening approaches and personalized therapeutic plans. A biomarker is a substance that can be measured and used as an indicator of a biological and/or pathological state. In cancer pathology, biomarkers can be used to detect the disease, to predict prognosis or response to therapy, and to evaluate the efficacy of therapy thereby improving the patient’s life expectancy and quality of life. The carcinoembryonic antigen (CEA) is the biomarker most frequently used in colorectal cancer. It is effective in the surveillance of colorectal cancer patients and in monitoring the efficacy of therapy, whereas it is less useful in colorectal screening. Recent studies indicate that also CEA-related proteins (CEACAMs) are promising potential colorectal cancer biomarkers. CEA and CEACAM proteins play important roles in cancer pathology, and the analysis of their functions will be useful for the identification of diagnostic and/or prognostic factors and therapeutic targets in colorectal and other cancers. List of Abbreviations CEA Carcinoembyonic Antigen CEACAM Carcinoembryonic Antigen-Family Cell Adhesion Molecule FOBT Fecal Occult Blood Test GPI Glycosylphosphatidylinositol Ig Immunoglobulin IgCAM Ig-like Cell Adhesion Molecule PCR Polymerase Chain Reaction TNM Tumor Nodes Metastases Key Facts of Colorectal Cancer • The intestine is the main site of nutrient absorption; it is subdivided in small and large intestine. The latter comprises the right colon, transverse colon, left colon, sigma, and rectum. • Colorectal cancer develops from uncontrolled growth of colorectal mucosa cells. • Colorectal cancer is an epithelial cancer arising in almost 80 % of cases from nonmalignant lesions that turned into cancer within 5–10 years of their appearance. 686 M. Gemei et al.

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.

Colorectal cancer (CRC) is one of the most common types of cancer; it is diagnosed in more than one million people each year and causes the death of about half of them. Early detection can help to decrease CRC-related mortality. In fact, the 5-year survival rate exceeds 90 % when the disease is localized and is only 10 % in case of metastases. Consequently, it is imperative to improve methods for the early detection of the disease. In this context, genomic approaches have resulted in new CRC biomarkers and have helped to shed light on the genetic basis of cancer as a whole. However, the proteome provides a more dynamic and faithful image of the genetic program of a cell. Hence, given the importance of proteins as effectors of cellular behavior, proteomic analysis has the potential to identify biomarkers that can help to classify and predict CRC. Several proteomic technique, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), quantitative isotopic protein labeling (SILAC, ICAT, and iTRAQ), and two-dimensional gel electrophoresis (2D-PAGE and 2D-DIGE), are being used in cancer research and can improve the diagnosis of patients; they are also helping to optimize personalized therapy. In this chapter, the main proteomic approaches used in biomarker research will be discussed, together with their impact and potential in the clinical setting. The proteomic biomarkers currently used for CRC diagnosis and/or therapy monitoring will be also described. List of Abbreviations 2D-DIGE Two-Dimensional Differential In-gel Electrophoresis 2D-PAGE Two-Dimensional Polyacrylamide Gel Electrophoresis APC Adenomatous Polyposis Coli CEA Carcinoembryonic Antigen CRC Colorectal Cancer Cy2 Cyanine 2 Fluorescent Dye Cy3 Cyanine 3 Fluorescent Dye Cy5 Cyanine 5 Fluorescent Dye DCBE Double-Contrast Barium Enema ESI Electrospray Ionization FIT Fecal Immunological Test FOBT Fecal Occult Blood Test *Email: salvator@unina.it Biomarkers in Cancer DOI 10.1007/978-94-007-7744-6_16-1 # Springer Science+Business Media Dordrecht 2014

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.

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.

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 106 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. Stem Cells 2014;32:1267–1277

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. © 2014 Springer Science+Business Media New York.

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.

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.

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 (CD66cbright) in CD133-positive cells from colon cancer samples. In vitro experiments demonstrated that colon spheres were considerably enriched in a CD66cbright 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: CD66cbright 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. Cancer 2013.

Recent findings suggested the presence in several cancers, comprising colorectal malignancies, of a small subset of tumor-initiating cells, or cancer stem cells. So the isolation and characterization of tumorigenic colorectal cancer cells was extremely important for the development of new diagnostic and therapeutic procedures. Even if a defined and universally approved phenotype of colorectal cancer stem cells has not been defined yet, several markers have been reported able to isolate cancer stem-like cells. For the majority of these markers, it has not been associated a clear function but more of them seem to be involved in colon cancer stem cell maintenance, survival and proliferation through well known pathways such as the Wnt-β-catenin pathway whose activity has been demonstrated to define colorectal cancer stem cells. Even if the knowledge on colorectal cancer stem cell “functioning” is still little detailed and elusive, the importance to develop new therapeutic strategies that precisely target colorectal cancer stem cells prompted researchers to test different approaches to this aim. We summarize here the phenotypic and functional characteristics associated with colon cancer stem cells along with the approaches experimented to selectively target and kill these cancer cells. We also reviewed innovative approaches targeting colorectal cancer stem cells based on new delivery systems to precisely kill them or also on the combination of conventional therapies with functional dietary elements.

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 subpopulation 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 up-regulation of the splicing factor SRp20, a newly identified target gene of the Wnt/??- 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 up-regulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.

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.

During the last decades, extended characterizations were performed of human full-term cord blood (hTCB) cells, but little information is available on human early preterm cord blood (hEPCB) hematopoietic stem cells (HSCs). In our study, we analyzed by flow cytometry 19 hEPCB and 17 hTCB samples. First, we observed that the percentage of CD34(Pos)CD45(Dim) cells was higher in hEPCB compared with hTCB and that it decreased during 16th-20th week of pregnancy. Within the CD34(Pos)CD45(Dim) population, we examined the expression of CD29, CD31, CD38, CD90, CD117, CD133, CD135, CD200, CD243, and CD338. We found that CD135 intensity and CD243(Pos) cells percentage were lower in hEPCB compared with hTCB. As to CD38, we observed that hEPCB samples were richer in undifferentiated CD34(Pos)CD45(Dim)CD38(Neg) HSCs compared with hTCB counterparts. We also compared the expression of the above-mentioned molecules in undifferentiated and committed HSCs residing in hEPCB and hTCB. In particular, although CD34(Pos)CD45(Dim)CD38(Pos) HSCs from both hEPCB and hTCB expressed relatively higher amounts of CD29, CD71, and CD135 compared with CD34(Pos)CD45(Dim)CD38(Neg) cells, a higher expression of CD31 was restricted to CD34(Pos)CD45(Dim)CD38(Pos) cells from hEPCB samples, and a higher expression of CD117 was demonstrated in CD34(Pos)CD45(Dim)CD38(Pos) cells from hTCB samples. Moreover, our data showed that CD34(Pos)CD45(Dim) cell population from hEPCB displayed higher percent of undifferentiated CD38(Neg)CD133(Pos) cells compared with hTCB samples. Finally, analyzing monocytes and lymphocytes within the two samples, we observed that T-cell percentages were higher in hTCB, whereas B-cell percentages were higher in hEPCB. We, therefore, studied the B-cell lineage maturation and found a higher percent of pro-B and pre-B cells in hEPCB compared with hTCB samples. Taken together, these results evidence the hematopoietic peculiarity of hEPCB, potentially useful for highlighting early steps of human hematolymphopoiesis as well as for developing novel strategies of stem cell-based therapy.

Mutations in the human parkin ( PARK2 ) gene cause autosomal recessive-juvenile Parkinson's disease (AR-JP). In Drosophila melanogaster , mutant parkin alleles display a broad range of phenotypic alterations, including female infertility. Here we report that reducing the level of eukaryotic translation initiation factor 4E ( eIF4E ) activity specifically rescues the female sterile phenotypes associated with the parkin P23 mutant allele. Additional defects, including reduction of pupal viability and body size, are also entirely recovered in both male and female flies of the abovementioned genotype. We further show that a null eIF4E-binding protein ( 4E-BP ) allele counteracts the in vivo effects produced, in a parkin P23 mutant background, by the reduction of functional eIF4E copy number. Moreover, Parkin and eIF4E interact in vitro and co-localize at the posterior end of developing oocytes. Finally, we show that eIF4E is over-expressed in parkin P23 mutant ovaries as compared to wild-types. Taken together, our data are consistent with the idea that Parkin and eIF4E act in a common pathway, likely modulating cap-dependent translation initiation events.

Background Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines. Methods MycoAlert® and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap®). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication. Results Mycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis. Conclusions Mycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression. In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.

International audience; Transformation to acute myeloid leukemia (AML) is a known complication of myeloproliferative neoplasia (MPN). Recent studies reported the high incidence (53%) of JAK2 negative blasts from transformed JAK2V617F-MPN. We collected, by cell sorting, blast cells and mature cells (GRA) from total bone marrow (BM) of 34 patients newly diagnosed of secondary AML. At MPN diagnosis (PMF n = 18; PV n = 9; ET n = 7), JAK2 was mutated in 22 of 34 patients. Twenty of 22 JAK2V617F-MPN (91%) maintained the mutation in blasts and GRA after leukemic switch, while in 2 of 22 patients the selected compartments lost the mutation. Surprisingly we also found the first case of JAK2V617F-AML from a wild type (WT)-MPN. In contrast to the previous study, we conclude that JAK2V617F-MPN yields rarely (9%) a JAK2WT-AML and any JAK2-status modification/persistence involves always the entire BM during leukemic transformation.

Abstract 480 Acute myeloid leukemia with mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features [Falini B et al, Blood 2007;109:874-885] which include negativity for CD34 antigen expression at both immunohistochemistry and gene expression profiling. Flow cytometric analysis shows that, in most NPM1-mutated AML, percentages of CD34+ cells are in the low range (< 5-10%). Detection of NPM1 mutations by molecular techniques and/or immunohistochemistry and Western Blot analysis with specific antibodies provides an important tool for tracking the genetic lesion in leukemic cells at different hierarchical stage. We previously reported involvement by NPM1 gene mutation of the CD34+ cell fraction isolated from patients with NPM1-mutated AML, and, in one case, the involvement, in particular, of the early progenitor CD34+/CD38- [Martelli MP et al, Blood (ASH Annual Meeting Abstracts) 2008;112:307]. Here we expand and confirm our previous observation in 5 cases of CD34-negative NPM1-mutated AML. CD34+/CD38- cells were isolated by either FACS (3 cases, purity >98%) or MACS-sorting (2 cases, purity >92%) and analyzed by molecular analysis and Western Blot with a specific anti-NPM1 mutant antibody, respectively. The presence of either NPM1 gene mutation or mutant protein was demonstrated in all samples analyzed proving the CD34+/CD38- cells belong to the leukemic clone. This cell subpopulation displayed also immunophenotypic features classically associated to leukemic stem cells (LSCs) (CD123+/CD33+/CD90-) in all (16/16) samples analyzed, suggesting they might actually represent the LSCs in NPM1-mutated AML. Indeed, CD34+ cell fraction isolated from NPM1-mutated AML was able to generate leukemia in immunocompromised mice resembling the original patient's disease. However, there is experimental evidence that, at least in some CD34-negative AML, also the CD34- population may contain LSCs. Whether the CD34- cell compartment in NPM1-mutated AML is also able to engraft and outgrow into leukemia in mice remains to be clarified. For this purpose, we assessed the engraftment ability of CD34- cells from 5 NPM1-mutated AML patients. No engraftment was observed in one case. Interestingly, in three patients with myelomonocytic (M4, 2 cases) and myelocytic (M2, 1 case) AML, the CD34- fraction resulted into marrow engraftment by human CD45+/CD33+ myeloid cells that, at morphological and immunohistological grounds, consisted of a mixed population of macrophage cells expressing the CD68 (PG-M1) antigen and mature looking myeloperoxidase (MPO)-positive cells. This pattern possibly reflects short-term engraftment by leukemic cells devoid of self-renewal potential that differentiated into mature elements. However, the neoplastic nature of engrafted cells could be established with certainty only in one case by western blotting detection of NPM1 mutant protein. Immunohistochemistry could not help in these cases to establish the leukemic nature of human cells since terminally differentiated leukemic cells in NPM1-mutated AML show nucleus-restricted NPM1 positivity. In contrast, the pure CD34+ fraction (availabel for comparison in one of these three cases) engrafted as AML with clear blastic morphology and cytoplasmic dislocation of nucleophosmin. In a fourth patient, the highly purified CD34- fraction from relapsed NPM1-mutated AML engrafted in mice with a typical AML picture. These preliminary findings suggest that in general the CD34- fraction from NPM1-mutated AML may have more limited engraftment potential than the CD34+ fraction. Further studies are ongoing to address this issue. Disclosures: Falini: Xenomics: Patents & Royalties.

Anabolic androgenic steroids, a class of steroid hormones related to testosterone, are natural ligands of androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. AR binds specific DNA elements, known as androgen-response elements. Testosterone, the main male sexual hormone, binds AR directly and indirectly, through conversion into dihydrotestosterone (DHT), its more active metabolite. Anabolic androgenic steroids are frequently detected in the urine of doped athletes; their consumption is also growing among sport amateurs and adolescents. The effects of androgens can differ depending on the target cells and/or tissues. To gain insight into transcription activation mechanisms of AR, we investigated AR protein signaling in human peripheral blood lymphocytes treated with supraphysiological doses of DHT. We performed a comparative proteomic analysis and we identified about 30 differentially expressed proteins. At least five species contained a consensus androgen-response elements sequence in the promoter region of related coding genes. The analysis also revealed that high doses of DHT activate the drug detoxification process, could stimulate an increase in cell motility and exert a prosurvival effect rather than an apoptotic one.

Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.

Background Mortality is high in patients with esophageal carcinoma as tumors are rarely detected before the disease has progressed to an advanced stage. Here, we sought to isolate cell-free DNA released into the plasma of patients with esophageal carcinoma, to analyze copy number variations of marker genes in the search for early detection of tumor progression. Methods Plasma of 41 patients with esophageal carcinoma was prospectively collected before tumor resection and chemotherapy. Our dataset resulted heterogeneous for clinical data, resembling the characteristics of the tumor. DNA from the plasma was extracted to analyze copy number variations of the erbB2 gene using real-time PCR assays. Results The real-time PCR assays for erbB2 gene showed significant (P = 0.001) copy number variations in the plasma of patients with esophageal carcinoma, as compared to healthy controls with high sensitivity (80%) and specificity (95%). These variations in erbB2 were negatively correlated to the progression free survival of these patients (P = 0.03), and revealed a further risk category stratification of patients with low VEGF expression levels. Conclusion The copy number variation of erbB2 gene from plasma can be used as prognostic marker for early detection of patients at risk of worse clinical outcome in esophageal cancer.

Abstract 4090 Transformation to acute myeloid leukemia (AML) is a known complication of MPN but the role of JAK2V617F mutation is still undefined. In 2006, Campbell described a possible model for the development of a JAK2WT-AML in a patient with JAK2V617F-MPN and more recently, Theocharides et al. reported that in up to 53% of the patients who developed secondary AML from a JAK2-mutated MPN the mutation was no longer detectable; however the results obtained with DNA extracted from cells scraped or laser-capture-microdissected from bone marrow (BM) or peripheral blood (PB) smears, were confirmed in fresh samples only in few cases. In this study, we collected, by cell sorting, blast cells and mature myeloid cells (granulocytes, GRA) from whole BM aspirates of 40 newly diagnosed patients with AML secondary to MPN (18 derived from PMF; 13 from PV and 9 from ET) and analyzed the JAK2 status before and after leukemic transformation in selected cell compartments. To evaluate the modification in the JAK2 status before and during leukemic transformation we performed ASO-PCR and (QRT)-PCR assay on total BM of the MPN phase and sorted cell populations from AML phase. At the time of MPN diagnosis, JAK2V617F was detectable in 28 of 40 patients (70%) (10 of 18 PMF; 12 of 13 PV and 6 of 9 ET). No cytogenetic abnormalities or MPL and JAK2-exone 12 mutations were detected at this stage. Median time to AML progression (TTP) was 5.09 years (yrs) (range 0.38 – 27.81). A significant difference (p= 0.02) in TTP was found grouping patients according to JAK2 status during the MPN phase [JAK2WT-MPN n=12, TTP median 15.10 yrs (0.38-16.32); JAK2 mutated-MPN n=28, TTP median 4.07 yrs (0.67-27.81)]. Eight patients showed additional abnormalities involving chromosomes 1, 5, 7, 8, 9, 12, 14, 17 and 20 while no other AML-associated mutations (FLT3, NPM, CEBPA, RUNX1) were detectable at this stage. In our cohort of patients we found that JAK2V617F mutation was still present at the blast transformation in both compartments: CD34+ cells (blasts) and CD15+ cells (GRA) in 24 of 28 JAK2 mutated MPN (86%). Four of 28 patients (14%) developed JAK2V617F negative AML starting from a mutated PV with a mean TTP of 5.14 yrs. Interestingly, the WT status was confirmed in blast cells but also in GRA. Surprisingly we found 2 cases of JAK2V617F mutated AML transforming from a WT-PMF. Also in this case the JAK2V617F positivity in the AML phase occurred in both GRA and blast compartments. No differences (p= 0.3) in the allele burden were found comparing MNCs from chronic phase with MNCs of leukemic transformations or comparing GRA with blasts in AML phase. In conclusion, these results contrast with the previous study in which the JAK2 mutation was lost in 53% of blasts during leukemia transformation. In our work, the loss of JAK2V617F mutation during AML progression is a rare event (14%). Additional studies in larger patient series and multivariate analysis are needed before a prognostic role of JAK2V617F mutation regarding time to leukemia transformation can be definitely assessed. Disclosures: Vannucchi: Novartis: Consultancy.

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.

// Francesca Puca 1 , Marianna Colamaio 1 , Antonella Federico 1 , Marica Gemei 2 , Nadia Tosti 1 , Andre Uchimura Bastos 1 , Luigi Del Vecchio 2 , Salvatore Pece 3 , Sabrina Battista 1 and Alfredo Fusco 1 1 Istituto di Endocrinologia ed Oncologia Sperimentale - CNR e/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita degli Studi di Napoli “Federico II”, Naples, Italy 2 CEINGE, Biotecnologie Avanzate, Naples, Italy 3 Istituto Europeo di Oncologia, Milan, Italy Correspondence: Alfredo Fusco, email: // Sabrina Battista, email: // Keywords : HMGA1, cancer stem cells, p53, colon carcinoma, NUMB Received : March 04, 2014 Accepted : April 16, 2014 Published : April 18, 2014 Abstract 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.