Elizabeth A. Butterworth

Asparagine synthetase: regulation by cell stress and involvement in tumor biology

Asparagine synthetase (ASNS) catalyzes the conversion of aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. The enzyme is ubiquitous in its organ distribution in mammals, but basal expression is relatively low in tissues other than the exocrine pancreas. Human ASNS activity is highly regulated in response to cell stress, primarily by increased transcription from a single gene located on chromosome 7. Among the genomic elements that control ASNS transcription is the C/EBP-ATF response element (CARE) within the promoter. Protein limitation or an imbalanced dietary amino acid composition activate the ASNS gene through the amino acid response (AAR), a process that is replicated in cell culture through limitation for any single essential amino acid. Endoplasmic reticulum stress also increases ASNS transcription through the PERK-eIF2-ATF4 arm of the unfolded protein response (UPR). Both the AAR and UPR lead to increased synthesis of ATF4, which binds to the CARE and induces ASNS transcription. Elevated expression of ASNS protein is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia and may be a predictive factor in drug sensitivity for certain solid tumors as well. Activation of the GCN2-eIF2-ATF4 signaling pathway, leading to increased ASNS expression appears to be a component of solid tumor adaptation to nutrient deprivation and/or hypoxia. Identifying the roles of ASNS in fetal development, tissue differentiation, and tumor growth may reveal that ASNS function extends beyond asparagine biosynthesis.

Transition from Colitis to Cancer: High Wnt Activity Sustains the Tumor-Initiating Potential of Colon Cancer Stem Cell Precursors

Ulcerative colitis (UC) increases the risk of colorectal cancer (CRC), but the mechanisms involved in colitis-to-cancer transition (CCT) are not well understood. CCT may involve a inflammation-dysplasia-carcinoma progression sequence compared with the better characterized adenoma-carcinoma progression sequence associated with sporadic CRC. One common thread may be activating mutations in components of the Wnt/β-catenin signaling pathway, which occur commonly as early events in sporadic CRC. To examine this hypothesis, we evaluated possible associations between Wnt/β-catenin signaling and CCT based on the cancer stem cell (CSC) model. Wnt/β-catenin immunostaining indicated that UC patients have a level of Wnt-pathway-active cells that is intermediate between normal colon and CRC. These UC cells exhibiting activation of the Wnt pathway constituted a major subpopulation (52% + 7.21) of the colonic epithelial cells positive for aldehyde dehydrogenase (ALDH), a putative marker of precursor colon CSC (pCCSC). We further fractionated this subpopulation of pCCSC using a Wnt pathway reporter assay. Over successive passages, pCCSCs with the highest Wnt activity exhibited higher clonogenic and tumorigenic potential than pCCSCs with the lowest Wnt activity, thereby establishing the key role of Wnt activity in driving CSC-like properties in these cells. Notably, 5/20 single cell injections of high-Wnt pCCSC resulted in tumor formation, suggesting a correlation with CCT. Attenuation of Wnt/β-catenin in high-Wnt pCCSC by shRNA-mediated downregulation or pharmacological inhibition significantly reduced tumor growth rates. Overall, the results of our study indicates (i) that early activation of Wnt/β-catenin signaling is critical for CCT and (ii) that high levels of Wnt/β-catenin signaling can further demarcate high-ALDH tumor-initiating cells in the nondysplastic epithelium of UC patients. As such, our findings offer plausible diagnostic markers and therapeutic target in the Wnt signaling pathway for early intervention in CCT.

ALDH as a marker for enriching tumorigenic human colonic stem cells.

Aldehyde dehydrogenase (ALDH) can be used as a marker to isolate, propagate, and track normal and cancerous human colon stem cells. To determine their tumorigenic potential, tissues obtained from proximal (normal counterpart) and distal (cancerous) colon of colon cancer patients are implanted into NOD-SCID mice. In parallel, ALDH(high) and ALDH(low) cells are isolated via Florescence Associated Cell Sorting (FACS) after the dissociation of distal and proximal colon tissues into a single-cell suspension. Flow cytometry for ALDH(high) and ALDH(low) cells is possible with the ALDEFLUOR assay. Following cell sorting, ALDH-enriched cells are tested for their tumorigenic potential in vivo as xenografts. Owing to cancer stem cell properties, ALDH(high) cells could be propagated in vivo by serial passaging of the human tissue as xenografts and in vitro as suspension cultures called sphere cultures. In this unit, all the above-mentioned methods to isolate and propagate colon cancer stem cells using ALDH as a stem cell marker are described in detail.

Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells.

Significance Cancer research has relied on in vitro experiments with established cell lines to investigate the efficacy of chemotherapy drug panels, which limit clinical correlation. Targeting tumor-initiating cells could be a viable clinical strategy, but these cells are extremely rare, necessitating new methods for rapid and robust screening. We developed a miniaturized platform to investigate the chemosensitivity of patient-derived tumor-initiating cells using limited cell numbers. The miniature platform described herein represents, to our knowledge, the first demonstration of a cell patterning technique that combines the ability to release small molecule drugs, alone or in combination, to separate islands of cells. This device can be adopted in clinical and academic laboratories targeting cancer stem cell populations to tailor patient-specific chemotherapeutic treatment options. A new paradigm in oncology establishes a spectrum of tumorigenic potential across the heterogeneous phenotypes within a tumor. The cancer stem cell hypothesis postulates that a minute fraction of cells within a tumor, termed cancer stem cells (CSCs), have a tumor-initiating capacity that propels tumor growth. An application of this discovery is to target this critical cell population using chemotherapy; however, the process of isolating these cells is arduous, and the rarity of CSCs makes it difficult to test potential drug candidates in a robust fashion, particularly for individual patients. To address the challenge of screening drug libraries on patient-derived populations of rare cells, such as CSCs, we have developed a drug-eluting microarray, a miniaturized platform onto which a minimal quantity of cells can adhere and be exposed to unique treatment conditions. Hundreds of drug-loaded polymer islands acting as drug depots colocalized with adherent cells are surrounded by a nonfouling background, creating isolated culture environments on a solid substrate. Significant results can be obtained by testing <6% of the cells required for a typical 96-well plate. Reliability was demonstrated by an average coefficient of variation of 14% between all of the microarrays and 13% between identical conditions within a single microarray. Using the drug-eluting array, colorectal CSCs isolated from two patients exhibited unique responses to drug combinations when cultured on the drug-eluting microarray, highlighting the potential as a prognostic tool to identify personalized chemotherapeutic regimens targeting CSCs.

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to <20 µm as formalin fixed paraffin section on glass slides or <500 µm for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples >500 µm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 µm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Islet Microvasculature Alterations With Loss of Beta-cells in Patients With Type 1 Diabetes:

Islet microvasculature provides key architectural and functional roles, yet the morphological features of islets from patients with type 1 diabetes are poorly defined. We examined islet and exocrine microvasculature networks by multiplex immunofluorescence imaging of pancreases from organ donors with and without type 1 diabetes (n=17 and n=16, respectively) and determined vessel diameter, density, and area. We also analyzed these variables in insulin-positive and insulin-negative islets of 7 type 1 diabetes donors. Control islet vessel diameter was significantly larger (7.6 ± 1.1 μm) compared with vessels in diabetic islets (6.2 ± 0.8 μm; p<0.001). Control islet vessel density (number/islet) was significantly lower (5.3 ± 0.6) versus diabetic islets (9.3 ± 0.2; p<0.001). Exocrine vessel variables were not significantly different between groups. Islets with residual beta-cells were comparable to control islets for both vessel diameter and density and were significantly different from insulin-negative islets within diabetic donors (p<0.05). Islet smooth muscle actin area had a significant positive correlation with age in both groups (p<0.05), which could negatively impact islet transplantation efficiency from older donors. These data underscore the critical relationship of islet beta-cells and islet vessel morphology in type 1 diabetes. These studies provide new knowledge of the islet microvasculature in diabetes and aging.

Abstract 107: p53 mutation at codon 273 in colorectal cancer and colitis-derived ALDHhigh cells promotes sphere formation in vitro

Introduction: p53 protein is a cell cycle regulator that functions as a tumor suppressor. Mutations in the p53 gene results in uncontrolled cell division, leading to diverse types of human cancer. In the case of colorectal cancer, p53 is the last gene to be mutated in the adenoma-to-carcinoma sequence. However, in colitis, early mutations in p53 occur in the course of dysplasia-to-carcinoma transition. Analysis of these mutations in colitis and colorectal cancer can provide clues regarding the pathogenesis of colitis to cancer.Recently, we have isolated and propagated ALDHhigh colon cancer stem cells (CCSCs) from colorectal cancer patients and precursor-CCSCs (pCCSCs) from colitic patients, which may be propagated as spheres. These CCSCs and pCCSCs may be used as a model system to study the transition from colitis to cancer. To determine if any of the p53 mutations correlate with sphere formation we analyzed p53 mutations in the existing sphere isolates. Methods: We performed sequence analysis of p53 mRNA and genomic DNA on 6 ALDHhigh sphere isolates derived from human colon cancer and 2 from colitic colon. Results: 5 out of 6 (83%) colorectal cancer derived ALDHhigh sphere isolates showed mutation at 273rd codon on the 8th exon (Arg→ His), which is one of the hot spots of p53 mutations in colorectal cancer. The same mutation was also observed in the ALDHhigh sphere isolates derived from colitic patients. This mutation corresponds to the amino acid residue that is in direct contact with DNA (contact mutants), but this mutation does not result in alterations in the spatial structure of p53. However, the defect in DNA binding ability of p53 allows the cell cycle to proceed unchecked and thus this mutation correlates with sphere forming ability. Conclusions: Mutations of p53 at codon 273 on exon 8 correlates with the sphere forming ability of the ALDHhigh cells derived from human colon cancer and colitic colon. Mutational knock-in and wild-type p53 overexpression studies would be vital to further confirm the role of this mutation in sphere formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 107. doi:1538-7445.AM2012-107

Abstract 5193: Wnt signaling in colitis derived colon cancer initiating cells promotes the tumorigenicity in colitis to cancer transition

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Wnt (Wingless) signaling in colon stem cells is involved in the development and maintenance of crypt architecture. In addition, activating mutations in the Wnt signaling pathway initiate and perpetuate colon cancer initiating cells (CCIC). Recently, we have isolated and propagated CCICs from colitis. Here, we hypothesize that Wnt signaling in the colitis derived CCICs potentiates colitis-associated tumor initiation. Methods: In order to test the above hypothesis, we have designed a dual fusion Wnt reporter construct (TTLG). The construct comprises of luminescent firefly luciferase and enhanced green fluorescent protein (eGFP), which are preceded by the TCF/LEF binding site. TTLG was transduced into colitis sphere isolates that were derived from Aldehyde dehydrogenase (ALDH) high cells. Immunohistochemistry validated the staining for activated β-Catenin in TTLG GFP high vs eGFP low colitis sphere isolates Tumorigenicity was evaluated by injecting eGFP high and eGFP low cells (1000 each) in the flanks of NOD-SCID mice. Secondary tumors were generated from 10% eGFP bright vs dimmest cells (1000, 100 and 10 each) obtained from eGFP high xenograft derived cells. In parallel, in vitro limiting dilution assays (LDA) were performed. Results: The eGFP high sphere cell population had nuclear localized Active Beta Catenin staining, indicating active Wnt signaling (95%). Conversely, little to no (<5%) Active Beta Catenin (ABC) staining was observed in the nucleus of eGFP low sphere cells. In vitro studies demonstrated that the clonogenic potential of eGFP high colitis sphere cells was significantly higher than that of eGFP low cells. This was further confirmed by in vivo studies, where the tumors generated from eGFP high cells grew more rapidly and larger when compared to eGFP low cells. The ability of the eGFP low cells to induce the tumor relates to the fact that eGFP low cells are a subset of the ALDH high population. Furthermore, secondary tumors that were generated from the 10% eGFP bright cells derived from eGFP high xenograft were initiated within few days after injection. At the time of harvest, the tumors formed from 10% eGFP dimmest cells were less than ¼ the size of the 10% eGFP bright cells derived tumor. The clonogenic potential of these 10% eGFP bright cells was about 50-100 fold higher than the 10% eGFP dimmest cells. Conclusions: The Wnt high ALDH high cells have higher clonogenic potential when compared to Wnt low ALDH high cells. Also, the tumors generated from Wnt high ALDH high colitis cells demonstrated decreased tumor latency and increased tumorigenicity compared to Wnt low ALDH high colitis cells. Collectively, our observations indicate that Wnt high CCICs from colitis promote colitis-associated tumorigenicity. 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 5193. doi:10.1158/1538-7445.AM2011-5193

Abstract 5189: An IL-8 dependent autocrine circuit promotes colorectal cancer tumorigenicity

Introduction: It is well established that chronic inflammation of the colon increases the incidence of colorectal cancer. One plausible mechanism involves inflammation-associated signals targeting the cancer stem cell (CSC). The CSC hypothesis posits that solid tumors originate from a rare cell population with stem cell-like properties, including self-renewal, and the ability to initiate tumor growth. We have previously demonstrated that CSC-like cells can be enriched from sporadic colorectal tumors (CRC) based on high level aldehyde dehydrogenase (ALDH) enzyme activity and expression of the epithelial cell adhesion molecule (EpCAM). The ALDH high /EpCAM + population was shown to initiate tumors in NOD-SCID mice, and termed the colon cancer initiating cell (CCIC). CRC-derived CCICs were able to be propagated in vitro under serum-free conditions as non-adherent spheres. IL-8, an inflammatory chemokine with known proliferative and angiogenic properties, is detected in the serum of patients with CRC. We have shown that antibodies targeting IL-8 inhibited the growth of CRC tumor xenografts. We hypothesize that an IL-8-dependent autocrine circuit promotes colorectal cancer tumorigenicity. Methods: Immunochemical analysis was used to detect expression of the IL8 receptors, CXCR1 and CXCR2, by CRC-derived spheres and tumor xenografts. IL-8 secretion by CRC-derived spheres was quantified by an IL-8 ELISA. To assess effects of IL-8 on tumor growth, continuous release pellets containing IL-8 (2 ng/ml) or a placebo were implanted into the flanks of NOD-SCID mice prior to injection of CRC-derived CCICs. Tumor volume was measured biweekly. Tumor vessel density was quantified by immunochemical detection of MECA-32 + blood vessels. To assess effects of IL-8 on CRC-derived CCIC sphere formation and growth, cells were plated in the presence or absence of exogenous IL-8 (0 to 20 ng/ml). Sphere number and size were enumerated on days 2 and 7. Results: Immunochemical analysis detected selective expression of the IL-8 receptor, CXCR1, by both CRC-derived spheres and xenografts. Significant amounts of IL-8 were secreted by CRC-derived spheres (3000 cells; 1513 pg/ml). The tumor growth rate of mice receiving an exogenous source of IL-8 was significantly higher than the placebo group (p=0.008). The resulting tumors exhibited an increased level of angiogenesis. The addition of increasing concentrations of exogenous IL-8 to CRC-derived CCIC cultures increased the sphere size at day 2 (10 and 20 ng/ml; p Conclusion: Collectively, these data suggest that colorectal cancer tumor initiation and growth can be modulated by the inflammation-associated chemokine IL-8 via an autocrine circuit. 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 5189. doi:10.1158/1538-7445.AM2011-5189