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The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs. Stem Cells 2015;33:35–44

Metabolism sustains the stemness of Cancer Stem Cells (CSCs), affecting, in turn, tumor heterogeneity, metastatic potential, and therapy resistance. Therefore, it is appealing to target CSCs metabolism as a new therapeutic approach. Consequently, we paid considerable attention to the anti-apoptotic microRNA miR-483-3p, that we reported being regulated by glucose metabolism in liver cancer cells. We investigated the therapeutic potential of targeting miR-483-3p by using the anti-glucose metabolism 2-deoxyglucose (2-DG) molecule in tumor Xenograft mouse model originating from two different Colon-Cancer Stem Cell lines (CCSC lines). We show that 2-DG treatment does not affect CCSCs during tumor formation in immunocompromised mouse models despite its ability to increase the CCSCs apoptotic rate in vitro and decrease miR-483-3p expression in both in vitro and in vivo experimental conditions. The promising in vitro data contrast with in vivo results that show the inefficacy of the treatment. We think that, in our immuno-compromised mouse model, to inhibit the glucose metabolism could become not only ineffective but also counterproductive by creating a selective pressure on the most fitting tumoral clones.

Despite advances in the curative approach, the survival rate of advanced colorectal cancer (CRC) patients is still poor, which is likely due to the emergence of cancer cell clones resistant to the available therapeutic options. We have already shown that CD44v6-positive CRC stem cells (CR-CSCs) are refractory toward standard anti-tumor therapeutic agents due to the activation of the PI3K pathway together with high HER2 expression levels. Tumor microenvironmental cytokines confer resistance to CR-CSCs against HER2/PI3K targeting by enhancing activation of the MAPK pathway. Here, we show that the CSC compartment, spared by BRAF inhibitor-based targeted therapy, is associated with increased expression levels of CD44v6 and Myc and retains boosted clonogenic activity along with residual tumorigenic potential. Inhibition of Myc transcription, downstream of the MAPK cascade components, and PI3K pathway activity was able to overcome the protective effects of microenvironmental cytokines, affecting the survival and the clonogenic activity of CR-CSCs, regardless of their mutational background. Likewise, the double targeting induced stabilization of mouse tumor avatars. Altogether, these data outline the rationale for dual kinase targeting of CR-CSCs to prevent their adaptive response, which would lead to disease progression.

Lipid Droplets (LDs) are emerging as crucial players in colon cancer development and maintenance. Their expression has been associated with high tumorigenicity in Cancer Stem Cells (CSCs), so that they have been proposed as a new functional marker in Colorectal Cancer Stem Cells (CR-CSCs). They are also indirectly involved in the modulation of the tumor microenvironment through the production of pro-inflammatory molecules. There is growing evidence that a possible connection between metabolic alterations and malignant transformation exists, although the effects of nutrients, primarily glucose, on the CSC behavior are still mostly unexplored. Glucose is an essential fuel for cancer cells, and the connections with LDs in the healthy and CSC populations merit to be more deeply investigated. Here, we showed that a high glucose concentration activated the PI3K/AKT pathway and increased the expression of CD133 and CD44v6 CSC markers. Additionally, glucose was responsible for the increased amount of Reactive Oxygen Species (ROS) and LDs in both healthy and CR-CSC samples. We also investigated the gene modulations following the HG treatment and found out that the healthy cell gene profile was the most affected. Lastly, Atorvastatin, a lipid-lowering drug, induced the highest mortality on CR-CSCs without affecting the healthy counterpart.

ObjectiveCancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy.DesignA collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance.ResultsHere we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy.ConclusionsWhile PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

Colorectal cancer is the fourth most common cause of cancer-related death. Although many advances in the treatment of this disease have been made, a large number of patients develop metastasis and resistance to current therapies. The current evidence indicates that cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) have crucial roles in colorectal carcinogenesis and metastasis. It is also very important to understand the mechanisms that allow the survival of CSCs, such as metabolic reprogramming, which permits them to obtain specific properties or the activation of alternative signaling pathways in response to first-line therapies. In this review, we discuss the failure of conventional therapies for colorectal cancer and provide a brief analysis of new therapeutic strategies for targeting non-responsive CSC. We highlight the use of combination therapies, such as horizontal or vertical targeting, as the most efficient strategy for eradicating these subpopulations of cancer cells.

// Bregje van Oorschot 1 , Giovanna Granata 1 , Simone Di Franco 2 , Rosemarie ten Cate 1 , Hans M. Rodermond 1 , Matilde Todaro 3 , Jan Paul Medema 1 , Nicolaas A.P. Franken 1 1 Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, Cancer Genomics Center, Amsterdam, The Netherlands 2 Department of Surgical, Oncological and Stomatological Sciences (DICHIRONS), Cellular and Molecular Pathophysiology Laboratory, University of Palermo, Palermo, Italy 3 Biomedical Department of Internal and Specialistic Medicine (DIBIMIS), University of Palermo, Palermo, Italy Correspondence to: Nicolaas A.P. Franken, email: n.a.franken@amc.uva.nl Keywords: radiation oncology, DNA repair, hyperthermia, double-strand breaks Received: May 10, 2016 Accepted: August 24, 2016 Published: September 01, 2016 ABSTRACT Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcs i ), we aim to enhance the effect of radiation. The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcs i [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcs i and HT were applied separately, and when both were combined, HT and DNA-PKcs i enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcs i compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcs i were found. In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcs i before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcs i to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

Cancer stem cells (CSCs) are considered to be the origin of cancer and it is suggested that they are resistant to chemotherapy. Current therapies fail to eradicate CSCs and therefore selecting a resistant cell subset that is able to facilitate tumor recurrences. Betulinic acid (BetA) is a broad acting natural compound, shown to induce cell death via the inhibition of the stearoyl-CoA- desaturase (SCD- 1). This enzyme converts saturated fatty acids into unsaturated fatty acids and is over-expressed in tumor cells. Here we show that BetA induces rapid cell death in all colon CSCs tested and is able to affect the CSCs directly as shown, via the loss of clonogenic capacity. Similar results were observed with inhibition of SCD-1, suggesting that SCD-1 activity is indeed a vulnerable link in colon CSCs and can be considered an ideal target for therapy in colon cancer.

ObjectiveCancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.DesignTo discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.ResultsThe drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.ConclusionsLY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

Colorectal tumours are actually considered as aberrant organs, within it is possible to notice a different stage of cell growth and differentiation. Their origin is reported to arise from a subpopulation of tumour cells endowed with, just like the healthy stem cells, self-renewal and aberrant multi-lineage differentiation capacity likely to be called colorectal cancer stem cells (CCSCs). Cancer stem cells (CSCs) fate, since their origin, reflects the influences from their microenvironment (or niche) both in the maintenance of stemness, in promoting their differentiation, and in inducing epithelial-mesenchymal transition, responsible of CSCs dissemination and subsequent formation of metastatic lesions. The tumour cells heterogeneity and their immuno-response resistance nowadays probably responsible of the failure of the conventional therapies, make this research field an open issue. Even more importantly, our increasing understanding of the cellular and molecular mechanisms that regulate CSC quiescence and cell cycle regulation, self-renewal, chemotaxis and resistance to cytotoxic agents, is expected to eventually result in tailor-made therapies with a significant impact on the morbidity and overall survival of colorectal cancer patients.

The mutual and interdependent interaction between tumor and its microenvironment is a crucial topic in cancer research. Recently, it was reported that targeting stromal events could improve efficacies of current therapeutics and prevent metastatic spreading. Tumor microenvironment is a "complex network" of different cell types, soluble factors, signaling molecules and extracellular matrix components, which orchestrate the fate of tumor progression. As by definition, cancer stem cells (CSCs) are proposed to be the unique cell type able to maintain tumor mass and survive outside the primary tumor at metastatic sites. Being exposed to environmental stressors, including reactive oxygen species (ROS), CSCs have developed a GSH-dependent antioxidant system to improve ROS defense capability and acquire a malignant phenotype. Nevertheless, tumor progression is dependent on extracellular matrix remodeling, fibroblasts and macrophages activation in response to oxidative stress, as well as epithelial mesenchymal transition (EMT)-inducing signals and endothelial and perivascular cells recruitment. Besides providing a survival advantage by inducing de novo angiogenesis, tumor-associated vessels contribute to successful dissemination by facilitating tumor cells entry into the circulatory system and driving the formation of pre-metastatic niche. In this review, we focus on the synergistic effect of hypoxia inducible factors (HIFs) and vascular endothelial growth factors (VEGFs) in the successful outgrowth of metastasis, integrating therefore many of the emerging models and theories in the field.

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the “differentiated” cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma–derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the “differentiated” tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

Tumor-initiating cells are responsible for tumor maintenance and relapse in solid and hematologic cancers. Although tumor-initiating cells were initially believed to be mainly quiescent, rapidly proliferating tumorigenic cells were found in breast cancer. In colon cancer, the proliferative activity of the tumorigenic population has not been defined, although it represents an essential parameter for the development of more effective therapeutic strategies. Here, we show that tumorigenic colon cancer cells can be found in a rapidly proliferating state in vitro and in vivo, both in human tumors and mouse xenografts. Inhibitors of polo-like kinase1 (Plk1), a mitotic kinase essential for cell proliferation, demonstrated maximal efficiency over other targeted compounds and chemotherapeutic agents in inducing death of colon cancer-initiating cells in vitro. In vivo, Plk1 inhibitors killed CD133+ colon cancer cells leading to complete growth arrest of colon cancer stem cell-derived xenografts, whereas chemotherapeutic agents only slowed tumor progression. While chemotherapy treatment increased CD133+ cell proliferation, treatment with Plk1 inhibitors eliminated all proliferating tumor-initiating cells. Quiescent CD133+ cells that survived the treatment with Plk1 inhibitors could be killed by subsequent Plk1 inhibition when they exited from quiescence. Altogether, these results provide a new insight into the proliferative status of colon tumor-initiating cells both in basal conditions and in response to therapy and indicate Plk1 inhibitors as potentially useful in the treatment of colorectal cancer.

INTRODUCTION: Recent evidence based on cancer stem cell (CSC) models, is boosting the progress of translational research and providing relevant clinical implications in many tumour types, including colorectal cancer. The current failure of standard therapies is attributed to a small fraction of the primary cell population with stem-like characteristics, such as self-renewal and differentiation. Identification of CSCs is based on two different criteria of selection: stemness-selective conditions and direct isolation based on putative stem cell markers expression. CD133, a transmembrane glycoprotein, was associated with tumor-initiating cells derived from several histological variants of tumors, including colon. AREAS COVERED: In this review the current understandings about CD133 as putative marker of tumour-initiating cells in colorectal cancer (CRC) is described. The focus of the discussion is on the need for additional markers to better identify the cell population able to recapitulate the parental tumor in immunocompromised mice. EXPERT OPINION: Identification and characterization of CSCs represents a relevant issue to define innovative therapeutic approaches, overcoming the emergence of cancer cell clones capable of evading standard therapy.

It is widely accepted by the scientific community that cancer, including colon cancer, is a “stem cell disease”. Until a few years ago, common opinion was that all neoplastic cells within a tumor contained tumorigenic growth capacity, but recent evidences hint to the possibility that such a feature is confined to a small subset of cancer-initiating cells, also called cancer stem cells (CSCs). Thus, malignant tumors are organized in a hierarchical fashion in which CSCs give rise to more differentiated tumor cells. CSCs possess high levels of ATP-binding cassette (ABC) transporters and anti-apoptotic molecules, active DNA-repair, slow replication capacities and they produce growth factors that confer refractoriness to antineoplastic treatments. The inefficacy of conventional therapies towards the stem cell population might explain cancer chemoresistance and the high frequency of relapse shown by the majority of tumors. Nowadays, in fact all the therapies available are not sufficient to cure patients with advanced forms of colon cancer since they target differentiated cancer cells which constitute most of the tumor mass and spare CSCs. Since CSCs are the entities responsible for the development of the tumor and represent the only cell population able to sustain tumor growth and progression, these cells represent the elective target for innovative therapies.