Catherine O'Brien

A human colon cancer cell capable of initiating tumour growth in immunodeficient mice

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells—cancer stem cells—possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 × 104 unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.

Cancer Stem Cells and Self-renewal

The cancer stem cell (CSC) or cancer-initiating cancer (C-IC) model has garnered considerable attention over the past several years since Dick and colleagues published a seminal report showing that a hierarchy exists among leukemic cells. In more recent years, a similar hierarchical organization, at the apex of which exists the CSC, has been identified in a variety of solid tumors. Human CSCs are defined by their ability to: (i) generate a xenograft that histologically resembles the parent tumor from which it was derived, (ii) be serially transplanted in a xenograft assay thereby showing the ability to self-renew (regenerate), and (iii) generate daughter cells that possess some proliferative capacity but are unable to initiate or maintain the cancer because they lack intrinsic regenerative potential. The emerging complexity of the CSC phenotype and function is at times daunting and has led to some confusion in the field. However, at its core, the CSC model is about identifying and characterizing the cancer cells that possess the greatest capacity to regenerate all aspects of the tumor. It is becoming clear that cancer cells evolve as a result of their ability to hijack normal self-renewal pathways, a process that can drive malignant transformation. Studying self-renewal in the context of cancer and CSC maintenance will lead to a better understanding of the mechanisms driving tumor growth. This review will address some of the main controversies in the CSC field and emphasize the importance of focusing first and foremost on the defining feature of CSCs: dysregulated self-renewal capacity. Clin Cancer Res; 16(12); 3113–20. ©2010 AACR.

Cancer Stem Cells in Solid Tumors: An Overview

It has long been appreciated that significant functional and morphologic heterogeneity can exist within the individual cells that comprise a tumor. Increasing evidence indicates that many solid tumors are organized in a hierarchical manner in which tumor growth is driven by a small subset of cancer stem cells (CSCs) or tumor-initiating cells. Although these cells represent a small percentage of the overall tumor population, they are the only cells capable of initiating and driving tumor growth. Emerging evidence indicates that these cells are also resistant to chemotherapy and radiation therapy, which has led to much speculation and interest surrounding the potential clinical applicability of CSCs.

ID1 and ID3 Regulate the Self-Renewal Capacity of Human Colon Cancer-Initiating Cells through p21

There is increasing evidence that some cancers are hierarchically organized, sustained by a relatively rare population of cancer-initiating cells (C-ICs). Although the capacity to initiate tumors upon serial transplantation is a hallmark of all C-ICs, little is known about the genes that control this process. Here, we establish that ID1 and ID3 function together to govern colon cancer-initiating cell (CC-IC) self-renewal through cell-cycle restriction driven by the cell-cycle inhibitor p21. Regulation of p21 by ID1 and ID3 is a central mechanism preventing the accumulation of excess DNA damage and subsequent functional exhaustion of CC-ICs. Additionally, silencing of ID1 and ID3 increases sensitivity of CC-ICs to the chemotherapeutic agent oxaliplatin, linking tumor initiation function with chemotherapy resistance.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large‐scale sequencing efforts. Using genome‐scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co‐culture competition assays to generate a high‐confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non‐isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN‐dependent genotypes across a series of non‐isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Colon cancer stem cells

This unit describes protocols for working with colon cancer stem cells. To work with these cells one must start by generating single-cell suspensions from human colon cancer tissue. These cell suspensions are sorted using flow cytometry-assisted cell sorting to fractionate the cells into tumor-initiating and nontumor-initiating subsets. Once the cells have been fractionated, they must be functionally tested to determine tumor-forming capacity, the gold standard being the in vivo xenograft assay. Methods have also been developed to grow these cells in vitro in a sphere-forming assay. This unit will describe how to isolate and functionally test colon cancer stem cells, as well as provide advice on the potential challenges of the research.

Surgeons' knowledge of quality indicators for gastric cancer surgery

BackgroundGastric cancer survival in the West is inferior to that achieved in Asian centers. While differences in tumor biology may play a role, poor quality surgery likely contributes to understaging. We hypothesize that the majority of surgeons performing gastric cancer surgery in North America are unaware of the recommended standards.MethodsUsing the Ontario College of Physicians and Surgeons registry, surgeons who potentially included gastric cancer surgery in their scope of practice were identified. A questionnaire was mailed to 559; of those, 206 surgeons reported managing gastric cancer. Results were evaluated by χ2 and logistic regression; P < 0.05 was considered significant.ResultsEighty-six percent of respondents were male and 53% practiced in an urban nonacademic setting. Forty percent reported operating on two to five cases of gastric cancer per year, and 42% on fewer than two cases per year. One-third of surgeons identified 4 cm or less to be the desired gross proximal margin. Half used frozen section to evaluate margin status. Twenty percent of surgeons were unsure of the number of lymph nodes (LN) needed to accurately stage gastric cancer, and the median number reported by the remainder was 10 (range, 0–30). Only 16 of 206 identified both a proximal margin of 5 cm or less and 15 or more LN as desired targets. Those performing more than five gastric resections per year were more likely to report a D2 resection (P = 0.008).ConclusionThe majority of surgeons operating on gastric cancer in Ontario did not identify recommended quality indicators of gastric cancer surgery. A continuing medical education program should be designed to address this knowledge gap to improve the quality of surgery and patient outcomes.

Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR

Combination therapy with EGFR inhibitors may overcome acquired resistance to PI3K pathway inhibitors in some colorectal cancer patients. Drug Resistance Reveals Vulnerability Kinase inhibitors are a common therapy for many cancers, but tumors frequently acquire resistance. Understanding the mechanisms of resistance can reveal new therapeutic options. Using various human and mouse models of KRAS-mutant colorectal cancer (CRC), Belmont et al. found that signaling by the receptor tyrosine kinase EGFR and the related ERBB family members was increased in CRC cells that had acquired resistance to a dual inhibitor of the kinases PI3K and mTOR. Increased EGFR expression was induced by the drug through the release of PI3K pathway–mediated inhibition of the transcription factor FOXO3a. Withdrawing the drug from culture medium returned EGFR abundance and signaling to pretreatment levels. Inhibiting EGFR restored sensitivity to the PI3K/mTOR inhibitor in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Thus, CRC patients that develop resistance to PI3K/mTOR inhibitors may benefit from additional treatment with EGFR inhibitors. Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC). We investigated potential bypass mechanisms to PI3K/mTOR inhibition in KRAS-mutant CRC. Using genetically engineered mouse model cells that had acquired resistance to the dual PI3K/mTOR small-molecule inhibitor PF-04691502, we determined with chemical library screens that inhibitors of the ERBB [epidermal growth factor receptor (EGFR)] family restored the sensitivity to PF-04691502. Although EGFR inhibitors alone have limited efficacy in reducing KRAS-mutant tumors, we found that PF-04691502 induced the abundance, phosphorylation, and activity of EGFR, ERBB2, and ERBB3 through activation of FOXO3a (forkhead box O 3a), a transcription factor inhibited by the PI3K to AKT pathway. PF-04691502 also induced a stem cell–like gene expression signature. KRAS-mutant patient-derived xenografts from mice treated with PF-04691502 had a similar gene expression signature and exhibited increased EGFR activation, suggesting that this drug-induced resistance mechanism may occur in patients. Combination therapy with dacomitinib (a pan-ERBB inhibitor) restored sensitivity to PF-04691502 in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Our findings suggest that combining PI3K/mTOR and EGFR inhibitors may improve therapeutic outcome in patients with KRAS-mutant CRC.

Administration of Hypoxia-Activated Prodrug Evofosfamide after Conventional Adjuvant Therapy Enhances Therapeutic Outcome and Targets Cancer-Initiating Cells in Preclinical Models of Colorectal Cancer

Purpose: Cancer-initiating cells (C-IC) have been described in multiple cancer types, including colorectal cancer. C-ICs are defined by their capacity to self-renew, thereby driving tumor growth. C-ICs were initially thought to be static entities; however, recent studies have determined these cells to be dynamic and influenced by microenvironmental cues such as hypoxia. If hypoxia drives the formation of C-ICs, then therapeutic targeting of hypoxia could represent a novel means to target C-ICs. Experimental Design: Patient-derived colorectal cancer xenografts were treated with evofosfamide, a hypoxia-activated prodrug (HAP), in combination with 5-fluorouracil (5-FU) or chemoradiotherapy (5-FU and radiation; CRT). Treatment groups included both concurrent and sequential dosing regimens. Effects on the colorectal cancer-initiating cell (CC-IC) fraction were assessed by serial passage in vivo limiting dilution assays. FAZA-PET imaging was utilized as a noninvasive method to assess intratumoral hypoxia. Results: Hypoxia was sufficient to drive the formation of CC-ICs and colorectal cancer cells surviving conventional therapy were more hypoxic and C-IC-like. Using a novel approach to combination therapy, we show that sequential treatment with 5-FU or CRT followed by evofosfamide not only inhibits tumor growth of xenografts compared with 5-FU or CRT alone, but also significantly decreases the CC-IC fraction. Furthermore, noninvasive FAZA-PET hypoxia imaging was predictive of a tumors response to evofosfamide. Conclusions: Our data demonstrate a novel means to target the CC-IC fraction by adding a HAP sequentially after conventional adjuvant therapy, as well as the use of FAZA-PET as a biomarker for hypoxia to identify tumors that will benefit most from this approach. Clin Cancer Res; 24(9); 2116–27. ©2018 AACR.

Abstract B01: Histone acetyltransferase 1 contributes to colon cancer initiating cell chemoresistance through DNA damage repair pathways

Despite advances in treatment, Colorectal cancer (CRC) remains a leading cause of cancer related deaths in North America. Most CRCs contain a subset of self-renewing colon cancer-initiating cells (CC-ICs) responsible for tumor initiation and maintenance. CC-ICs are relatively chemoresistant as compared to the bulk tumor cells, however the mechanisms of this chemoresistance are poorly understood. An increasing amount of evidence suggests that epigenetic regulators may be playing a central role in driving CC-IC chemoresistance; one such example being histone acetyltransferase (HAT1), which plays an important role in cancer cell proliferation and response to DNA damage (Xue et al, Int J Clin Exp Pathol, 2014). The cytoplasmic HAT1/HAT2 complex acetylates histone H4 on lysine residues 5 and/or 12. Acetylated histone H4 is then bound to histone H3 and the complex is transported to the nucleus where the HAT1/HAT2 complex aids in depositing H3/H4 onto DNA (Parthum, Oncogene, 2007). In normal colon, HAT1 is predominantly localized to the nucleus in cells at the crypt base, whereas in primary and metastatic colorectal tumors, HAT1 expression is upregulated, mostly cytoplasmic, and diffuse throughout the tissue (Seiden-Long et al, Oncogene, 2006). To investigate HAT1 localization in our primary derived CC-IC enriched cell lines we used protein fractionation and western blot analysis. In our samples, we consistently see elevated cytoplasmic HAT1 expression compared to nuclear extracts. To determine the effect of the loss of HAT1 on CC-IC viability we infected our CC-IC enriched cell lines with lentiviral shRNA to knockdown HAT1. We observed no changes in growth or cell viability at baseline between the HAT1 knockdown cells and controls, however, previous studies have shown that HAT1 knockdown increases cancer cell sensitivity to DNA damaging agents such as camptothecin (CPT) or methyl methane sulfonate (MMS) by controlling Rad52 degradation, accumulating replication block double stranded breaks and prolonging G2 arrest, indicating that it is important for DNA repair processing (Barman et al, BBRC, 2006). To determine whether HAT1 contributes to CC-IC chemoresistance we treated our HAT1 knockdown cells with standard of care (SOC) chemotherapy (oxaliplatin). Viability assays showed that after 10 days of treatment there is a 40% decrease in cell viability compared to controls. To investigate whether the decrease in cell viability seen in HAT1 knockdown cells is due to an increase in DNA damage we stained cells for DNA damage marker γH2AX. Immunofluorescence analysis revealed a higher level of γH2AX staining in HAT1 knockdown cells treated with SOC chemotherapy. To examine the effects of HAT1 knockdown on CC-IC function when combined with SOC chemotherapy, we preformed a limiting dilution assay. Untreated CC-IC enriched HAT1 knockdown cells had no significant difference in CC-IC frequency, however, treating these cells with SOC chemotherapy resulted in a decrease in CC-IC frequency in vitro (212.2 vs 52.8). Our current research suggests that HAT1 plays an important role in CC-IC function, chemoresistance and response to DNA damage. We are further investigating HAT1 as a potential therapeutic target in order to improve outcomes for CRC patients. Citation Format: Lauren Agro, Cherry Leung, Yadong Wang, Evelyne Lima-Fernandes, Catherine O9Brien. Histone acetyltransferase 1 contributes to colon cancer initiating cell chemoresistance through DNA damage repair pathways [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B01.