Federico Tozzi

Endothelial Cells Promote the Colorectal Cancer Stem Cell Phenotype through a Soluble Form of Jagged-1

We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive CRC cells colocalized in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC-conditioned medium or blockade of ADAM17 activity. Collectively, ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Overexpression of Snail induces epithelial- mesenchymal transition and a cancer stem cell- like phenotype in human colorectal cancer cells

Epithelial–mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC‐like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail‐overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC‐like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.

Identification of cancer stem cells in human gastrointestinal carcinoid and neuroendocrine tumors

BACKGROUND & AIMS Metastatic gastrointestinal neuroendocrine tumors (NETs) frequently are refractory to chemotherapy. Chemoresistance in various malignancies has been attributed to cancer stem cells (CSCs). We sought to identify gastrointestinal neuroendocrine CSCs (N-CSCs) in surgical specimens and a NET cell line and to characterize novel N-CSC therapeutic targets. METHODS Human gastrointestinal NETs were evaluated for CSCs using the Aldefluor (Stemcell Technologies, Vancouver, Canada) assay. An in vitro, sphere-forming assay was performed on primary NET cells. CNDT2.5, a human midgut carcinoid cell line, was used for in vitro (sphere-formation) and in vivo (tumorigenicity assays) CSC studies. N-CSC protein expression was characterized using Western blotting. In vivo, systemic short interfering RNA administration targeted Src. RESULTS By using the Aldefluor assay, aldehyde dehydrogenase-positive (ALDH+) cells comprised 5.8% ± 1.4% (mean ± standard error of the mean) of cells from 19 patient samples. Although many primary cell lines failed to grow, CNDT96 ALDH+ cells formed spheres in anchorage-independent conditions, whereas ALDH- cells did not. CNDT2.5 ALDH+ cells formed spheres, whereas ALDH- cells did not. In vivo, ALDH+ CNDT2.5 cells generated more tumors, with shorter latency than ALDH- or sham-sorted cells. Compared with non-CSCs, ALDH+ cells demonstrated increased expression of activated Src, Erk, Akt, and mammalian target of rapamycin (mTOR). In vivo, anti-Src short interfering RNA treatment of ALDH+ tumors reduced tumor mass by 91%. CONCLUSIONS CSCs are present in NETs, as shown by in vitro sphere formation and in vivo tumorigenicity assays. Src was activated in N-CSCs and represents a potential therapeutic target in gastrointestinal NETs.

Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells

Background:Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells.Methods:Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells.Results:Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance.Conclusion:Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells

Background:Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs.Methods:Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment.Results:None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not.Conclusions:PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

ATP Citrate Lyase Mediates Resistance of Colorectal Cancer Cells to SN38

Combination chemotherapy is standard for metastatic colorectal cancer; however, nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may enable identification of novel targets and more effective therapy. Irinotecan is commonly used in first- and second-line therapy for patients with metastatic colorectal cancer, with the active metabolite being SN38. Emerging evidence suggests that altered metabolism in cancer cells is fundamentally involved in the development of drug resistance. Using Oncomine and unbiased proteomic profiling, we found that ATP citrate lyase (ACLy), the first-step rate-limiting enzyme for de novo lipogenesis, was upregulated in colorectal cancer compared with its levels in normal mucosa and in chemoresistant colorectal cancer cells compared with isogenic chemo-naïve colorectal cancer cells. Overexpression of exogenous ACLy by lentivirus transduction in chemo-naïve colorectal cancer cells led to significant chemoresistance to SN38 but not to 5-fluorouracil or oxaliplatin. Knockdown of ACLy by siRNA or inhibition of its activity by a small-molecule inhibitor sensitized chemo-naïve colorectal cancer cells to SN38. Furthermore, ACLy was significantly increased in cancer cells that had acquired resistance to SN38. In contrast to chemo-naïve cells, targeting ACLy alone was not effective in resensitizing resistant cells to SN38, due to a compensatory activation of the AKT pathway triggered by ACLy suppression. Combined inhibition of AKT signaling and ACLy successfully resensitized SN38-resistant cells to SN38. We conclude that targeting ACLy may improve the therapeutic effects of irinotecan and that simultaneous targeting of ACLy and AKT may be warranted to overcome SN38 resistance. Mol Cancer Ther; 12(12); 2782–91. ©2013 AACR.

Abstract B64: Development of a novel strategy to overcome drug resistance by targeting ATP citrate lyase and de novo lipogenesis in colorectal cancer cells

Background: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Although the response rate to current systemic therapies is ∼50%, drug resistance develops in nearly all patients leading to 50,000 deaths each year. Overcoming drug resistance involves understanding the mechanisms by which cancer cells adapt to the genotoxic stress. Metabolic changes at levels of mitochondria function, glucose metabolism, and de novo synthesis of fatty acids frequently occurs in malignant cells and impacts tumor development and growth. Our laboratory established in vitro drug-resistant models of CRC cells by chronic exposure of HT29 cells to increasing doses of chemotherapeutic agents (oxaliplatin, 5-FU and SN38) over a period of 4–6 months. We recently reported a metabolic switch to the glycolytic phenotype due to mitochondria defects in the oxaliplatin-resistant CRC cells (HT29-OXR) (Zhou et al, Cancer Research, 2012). In this study, we tested the hypothesis that deregulation of de novo lipogenesis pathways plays an important role in chemoresistance of CRC cells. Methods: A previous study (Bose et al. Br J Ca, 2011) using unbiased proteomic profiling by mass spectroscopy (MS) was used to determine the proteomic signature of defective metabolic pathways in the oxaliplatin-resistant CRC cells (OXR cells). ATP-citrate-lyase (ACLy), the key enzyme of de novo lipogenesis pathway, was examined for protein levels and activation. The lipid content of resistant cells was examined by transmission electron microscope (TEM) and Oil Red staining. Transient knockdown of the ACLy protein by siRNA was used to study re-chemosensitization of the resistant cells. A small molecule inhibitor of ACLy was studied in combination with chemotherapeutic agents in resistant CRC cells for growth inhibitory (MTT assay) and cytotoxic effects (PARP cleavage and Annexin V staining). A cell free assay was used to test the potency of the ACLy inhibitor on ACLy activation. Results: Activated ACLy protein level (phosphorylation on S454) was demonstrated by Western blot analysis in the HT29-OXR and SN38-resistant (HT29-SNR) cells, but not in 5-FU-resistant (HT29-FUR) cells. The OxR cells showed a 2–3 fold increase in lipid droplets numbers (by TEM examination) and fatty acid content (by Oil Red staining) than the parental cells. Furthermore, transient knockdown of the ACLy protein by siRNA demonstrated a return to chemosensitization when cells were treated with oxaliplatin. IC50 values of the ACLy inhibitor for parental HT29 and HT29-OXR, -SNR and —FUR cells were ∼30μM. As a single agent, the ACLy inhibitor blocked phosphorylation of ACLy and induced apoptosis in a concentration-dependent manner in parental HT29 cells, and its resistant derivatives -OXR and -SNR cells. Combination of the ACLy inhibitor at concentrations sufficient to block ACLy phosphorylation with oxaliplatin and SN38 showed enhanced effects on growth inhibition (MTT) and apoptosis induction (PARP cleavage and Annexin V assay) in HT29 cells-OXR and -SNR cells. Conclusions: Chemoresistant CRC cells demonstrated: 1) increased de novo lipogenesis, 2) elevated levels of key lipogenesis enzymes ACLy, 3) dependence on ACLy activity for cell survival under cytotoxic stress. This metabolic switch likely contributes to the chemoresistant phenotype of CRC cells. Targeting an early step of de novo lipogenesis such as blocking ACLy activity may provide a novel strategy to overcome drug-resistance in CRC cells.

Abstract 5132: Hypo-glycosylation of vascular endothelial growth factor receptor-1 may contribute to intracrine signaling in human colorectal cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Vascular endothelial growth factor receptor-1 (VEGFR-1) is highly expressed in endothelial cells, macrophages and cancer cells in the tumor microenvironment. VEGFR-1 plays an important role in tumor angiogenesis, inflammation and metastasis. In colorectal cancer (CRC) cells with hyperactive Wnt signaling, its function has been linked to cell motility, anchorage-independent growth, and cell survival. To better understand the role of VEGFR-1 in CRC, we performed studies to define the subcellular location and post-translational modifications of VEGFR-1 in human CRC cells. Methods/Results: Utilizing IHC analysis, GFP-tagged imaging and cell surface receptor assays, we observed that VEGFR-1 locates mostly in intracellular compartments in CRC cells, which is distinct from its membrane receptor status in endothelial cells. Moreover, spontaneous apoptosis was increased with endogenous VEGF knockout whereas treatment with the VEGF neutralizing antibody bevacizumab had no effect on apoptosis (Samuel et al. Oncogene, in press); these studies suggest that CRC cells require VEGF/VEGFR-1 intracrine signaling for suppression of apoptosis. In order to understand the means by which VEGFR-1 cellular localization is determined, we analyzed the glycosylation of VEGFR-1 in CRC cells, endothelial cells and clinical specimens of metastatic CRC from the liver. We found that VEGFR-1 is partially N-glycosylated by high mannose oligosaccharides in the CRC cells and human tumors, in contrast to complex, mature N-glycosylation of VEGFR-1 in the endothelial cells and normal liver tissue. Conclusion: VEGFR-1 is differentially post-translationally modified and intracellularly localized in CRC cells and metastatic tumors. Because N-glycosylation is critical for the membrane receptors intracellular trafficking and ligand interaction on the cell surface, hypo-glycosylation of VEGFR-1 in CRC cells likely contributes to its cytoplasmic localization and intracrine activation through a non-RTK pathway. The implications of intracrine function of VEGFR-1 in CRC warrant further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5132. doi:10.1158/1538-7445.AM2011-5132

Abstract 4075: Chemoresistant colorectal cancer cells exhibit high glycolytic activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Overcoming drug resistance in colorectal cancer (CRC) requires an understanding the mechanisms by which cancer cells adapt to the genotoxic stress. We established oxaliplatin resistant CRC cells derived from HCT116 and HT29 cell lines (HCT116 OxR and HT29 OxR) (Yang AD et al, Clin Ca Res, 2007). The mechanisms by which these resistant clones reprogram their energy metabolism to gain a survival advantage remain unknown. In this study, we hypothesized that chemoresistant CRC cells would exhibit altered cellular metabolism in order to survive following chronic genotoxic stress. Methods: The expression level of the glycolytic enzymes Glut1, hexokinase II (HK2), LDHA and HIF1α were detected by Western Blotting. Glucose uptake and lactate production was calculated by measuring the concentration of glucose and lactate in the culture media. Intracellular ATP/ADP levels, oxygen consumption and mitochondria ATP production were quantified. An in vivo xenograft study was used to compare the growth rate and angiogenesis of parental and oxaliplatin-resistant cells. Results: Compared with the parental cells, both HT29-OxR and HCT116-OxR cells exhibited a metabolic phenotype with increased glycolysis as reflected by increased glucose uptake and lactate production. Glycolytic enzymes were upregulated in resistant cells including Glut1, HK2 and LDHA. HIF1α expression and VEGF levels in the conditioned media were increased in the resistant cells. The mitochondria of OxR cells demonstrated defective complex I/II substrate ATP production despite increased cellular oxygen consumption. Importantly, the OxR cells maintain higher levels of intracellular ATP and ATP/ADP ratio indicating a metabolic switch to glycolysis. In a tumor xenograft model, HT29 OxR cells grew significantly slower than parental HT29 cells. Interestingly, when OxR cells were mixed with parental HT29 cells (50:50 and 90:10), tumor growth and microvessel counts were significantly increased. Conclusion: Oxaliplatin-resistant cells demonstrated: 1) high aerobic glycolytic activity, 2) an increase in HIF1α and glycolytic enzymes, and 3) defective mitochondria function. This metabolic switch provides more ATP production and likely contributes to the chemoresistant phenotype. Altering energy metabolism may provide a novel strategy to overcome drug-resistance in CRC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2011-4075

Abstract 5199: Primary culture of human midgut carcinoid cell lines and isolation of a putative carcinoid stem cell population

Background: Research in carcinoid and neuroendocrine tumors (NETs) of midgut origin is limited by a lack of cell lines. The existing foregut NET lines, BON and H727, are phenotypically distinct from midgut NETs highlighting the importance of establishing midgut derived NET cell lines. Therapeutic options are limited for midgut NETs. NET cell lines are difficult to culture for long periods (thus few cell lines). We are developing methods to improve primary and prolonged NET cell culture in an attempt to identify novel therapeutic approaches. Along these lines, we are conducting studies to identify a putative cancer stem cell population in human NET specimens to provide new insights regarding potential targets in NETs. Methods: Sterile human midgut NET surgical specimens were digested to a single cell suspension and fibroblast depleted with magnetic beads. The NET cells were maintained on collagen I coated plates for primary culture. A portion of the primary cell culture was used for FACS by the Aldefluor assay to isolate potential cancer stem cells (CSCs). Sorted cells were grown in serum-free media on low attachment plates to assess sphere formation and preserved for RNA isolation and subsequent gene array. Subcutaneous xenografts in nude and NOD/SCID gamma mice using fresh tumor implants and NET cell suspensions were performed as potential means to further propagate NET cells. Results: The NET markers, somatostatin receptor 2 and chromogranin A, were detectable in cultures from a midgut carcinoid liver metastasis at passage 3 and 5, as well as in one mouse xenograft. While retention of NET markers from xenograft implants is promising, tumor growth remains limited and slow, with tumors in 5/59 mice over 4-10 months. We are now studying tumor xenografts in more immunodeficient NOD/SCID gamma mice with current patient samples to potentially enhance xenograft growth. FACS sorting identified an ALDH+ population of NET cells, which formed spheres (a characteristic of CSCs) more frequently than ALDH(−) negative cells. A FACS sorted midgut sample plated at 10k cells/well in sphere forming media produced spheres as early as 8 days in the ALDH+ population, while the ALDH- cells did not form spheres (sham sorted cells formed rare spheres), which was also observed in other samples where viable cells were obtained. Additionally, the ALDH+ cells from this population were cutlured on plastic and remained viable for 4 weeks, while the ALDH- population was not viable in culture. RNA isolation for gene array is underway for the ALDH+ population to characterize these cells in midgut carcinoids. Conclusions: We have developed a system allowing for NET primary culture for up to 4 weeks. We have also identified a putative CSC population in NETs. These techniques are the first step in obtaining cells for in vitro studies and in identifying novel therapeutic targets for NETs. Supported by the The Raymond and Beverly Sackler Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2011-5199