Dale W. Lewis

A Stochastic Model for Cancer Stem Cell Origin in Metastatic Colon Cancer

Human cancers have been found to include transformed stem cells that may drive cancer progression to metastasis. Here, we report that metastatic colon cancer contains clonally derived tumor cells with all of the critical properties expected of stem cells, including self-renewal and the ability to differentiate into mature colon cells. Additionally, when injected into mice, these cells initiated tumors that closely resemble human cancer. Karyotype analyses of parental and clonally derived tumor cells expressed many consistent (clonal) along with unique chromosomal aberrations, suggesting the presence of chromosomal instability in the cancer stem cells. Thus, this new model for cancer origin and metastatic progression includes features of both the hierarchical model for cancerous stem cells and the stochastic model, driven by the observation of chromosomal instability.

Deficiency in myosin light-chain phosphorylation causes cytokinesis failure and multipolarity in cancer cells

Cancer cells often have unstable genomes and increased centrosome and chromosome numbers, which are an important part of malignant transformation in the most recent model of tumorigenesis. However, very little is known about divisional failures in cancer cells that may lead to chromosomal and centrosomal amplifications. In this study, we show that cancer cells often failed at cytokinesis because of decreased phosphorylation of the myosin regulatory light chain (MLC), a key regulatory component of cortical contraction during division. Reduced MLC phosphorylation was associated with high expression of myosin phosphatase and/or reduced myosin light-chain kinase levels. Furthermore, expression of phosphomimetic MLC largely prevented cytokinesis failure in the tested cancer cells. When myosin light-chain phosphorylation was restored to normal levels by phosphatase knockdown, multinucleation and multipolar mitosis were markedly reduced, resulting in enhanced genome stabilization. Furthermore, both overexpression of myosin phosphatase or inhibition of the myosin light-chain kinase in nonmalignant cells could recapitulate some of the mitotic defects of cancer cells, including multinucleation and multipolar spindles, indicating that these changes are sufficient to reproduce the cytokinesis failures we see in cancer cells. These results for the first time define the molecular defects leading to divisional failure in cancer cells.

The p53–PUMA axis suppresses iPSC generation

Mechanisms underlying the reprogramming process of induced pluripotent stem cells remain poorly defined. Like tumorigenesis, generation of induced pluripotent stem cells was shown to be suppressed by the Trp53 (p53) pathway, at least in part via p21Cdkn1a (p21)-mediated cell cycle arrest. Here we examine the role of PUMA, a pro-apoptotic mediator of p53, during somatic reprogramming in comparison to p21 in the p53 pathway. Using mouse strains deficient in these molecules, we demonstrate that PUMA is an independent mediator of the negative effect of p53 on induced pluripotent stem cell induction. PUMA deficiency leads to a better survival rate associated with reduced DNA damage and fewer chromosomal aberrations in induced pluripotent stem cells, whereas loss of p21 or p53 results in an opposite outcome. Given these new findings, PUMA may serve as a distinct and more desirable target in the p53 pathway for induced pluripotent stem cell generation, thereby having important implications for potential therapeutic applications of induced pluripotent stem cells.

Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+ cells.

Etoposide is a widely used anticancer drug successfully used for the treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia involving the mixed-lineage leukemia (MLL) gene (11q23) translocations. Previous studies demonstrated that the phenoxyl radical of etoposide can be produced by action of myeloperoxidase (MPO), an enzyme found in developing myeloid progenitor cells, the likely origin for myeloid leukemias. We hypothesized, therefore, that one-electron oxidation of etoposide by MPO to its phenoxyl radical is important for converting this anticancer drug to genotoxic and carcinogenic species in human CD34+ myeloid progenitor cells. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34+ cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In separate studies, etoposide-induced DNA damage and MLL gene rearrangements were demonstrated to be dependent in part on MPO activity in CD34+ cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34+ cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia.

Targeted inhibition of ATR or CHEK1 reverses radioresistance in oral squamous cell carcinoma cells with distal chromosome arm 11q loss.

Oral squamous cell carcinoma (OSCC), a subset of head and neck squamous cell carcinoma (HNSCC), is the eighth most common cancer in the U.S.. Amplification of chromosomal band 11q13 and its association with poor prognosis has been well established in OSCC. The first step in the breakage‐fusion‐bridge (BFB) cycle leading to 11q13 amplification involves breakage and loss of distal 11q. Distal 11q loss marked by copy number loss of the ATM gene is observed in 25% of all Cancer Genome Atlas (TCGA) tumors, including 48% of HNSCC. We showed previously that copy number loss of distal 11q is associated with decreased sensitivity (increased resistance) to ionizing radiation (IR) in OSCC cell lines. We hypothesized that this radioresistance phenotype associated with ATM copy number loss results from upregulation of the compensatory ATR‐CHEK1 pathway, and that knocking down the ATR‐CHEK1 pathway increases the sensitivity to IR of OSCC cells with distal 11q loss. Clonogenic survival assays confirmed the association between reduced sensitivity to IR in OSCC cell lines and distal 11q loss. Gene and protein expression studies revealed upregulation of the ATR‐CHEK1 pathway and flow cytometry showed G2M checkpoint arrest after IR treatment of cell lines with distal 11q loss. Targeted knockdown of the ATR‐CHEK1 pathway using CHEK1 or ATR siRNA or a CHEK1 small molecule inhibitor (SMI, PF‐00477736) resulted in increased sensitivity of the tumor cells to IR. Our results suggest that distal 11q loss is a useful biomarker in OSCC for radioresistance that can be reversed by ATR‐CHEK1 pathway inhibition.

Upregulation of the ATR-CHEK1 pathway in oral squamous cell carcinomas

The ATR‐CHEK1 pathway is upregulated and overactivated in Ataxia Telangiectasia (AT) cells, which lack functional ATM protein. Loss of ATM in AT confers radiosensitivity, although ATR‐CHEK1 pathway overactivation compensates, leads to prolonged G2 arrest after treatment with ionizing radiation (IR), and partially reverses the radiosensitivity. We observed similar upregulation of the ATR–CHEK1 pathway in a subset of oral squamous cell carcinoma (OSCC) cell lines with ATM loss. In the present study, we report copy number gain, amplification, or translocation of the ATR gene in 8 of 20 OSCC cell lines by FISH; whereas the CHEK1 gene showed copy number loss in 12 of 20 cell lines by FISH. Quantitative PCR showed overexpression of both ATR and CHEK1 in 7 of 11 representative OSCC cell lines. Inhibition of ATR or CHEK1 with their respective siRNAs resulted in increased sensitivity of OSCC cell lines to IR by the colony survival assay. siRNA‐mediated ATR or CHEK1 knockdown led to loss of G2 cell cycle accumulation and an increased sub‐G0 apoptotic cell population by flow cytometric analysis. In conclusion, the ATR‐CHEK1 pathway is upregulated in a subset of OSCC with distal 11q loss and loss of the G1 phase cell cycle checkpoint. The upregulated ATR‐CHEK1 pathway appears to protect OSCC cells from mitotic catastrophe by enhancing the G2 checkpoint. Knockdown of ATR and/or CHEK1 increases the sensitivity of OSCC cells to IR. These findings suggest that inhibition of the upregulated ATR–CHEK1 pathway may enhance the efficacy of ionizing radiation treatment of OSCC.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66- is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance.

Cell division patterns and chromosomal segregation defects in oral cancer stem cells.

Oral squamous cell carcinoma (OSCC) is a serious public health problem caused primarily by smoking and alcohol consumption or human papillomavirus. The cancer stem cell (CSC) theory posits that CSCs show unique characteristics, including self‐renewal and therapeutic resistance. Examining biomarkers and other features of CSCs is critical to better understanding their biology. To this end, the results show that cellular SOX2 immunostaining correlates with other CSC biomarkers in OSCC cell lines and marks the rare CSC population. To assess whether CSC division patterns are symmetrical, resulting in two CSC, or asymmetrical, leading to one CSC and one cancer cell, cell size and fluorescence intensity of mitotic cells stained with SOX2 were analyzed. Asymmetrical SOX2 distribution in ≈25% of the mitoses analyzed was detected. Chromosomal instability, some of which is caused by chromosome segregation defects (CSDs), is a feature of cancer cells that leads to altered gene copy numbers. We compare chromosomal instability (as measured by CSDs) between CSCs (SOX2+) and non‐CSCs (SOX2−) from the same OSCC cell lines. CSDs were more common in non‐CSCs (SOX2−) than CSCs (SOX2+) and in symmetrical CSC (SOX2+) mitotic pairs than asymmetrical CSC (SOX2+/SOX2−) mitotic pairs. CSCs showed fewer and different types of CSDs after ionizing radiation treatment than non‐CSCs. Overall, these data are the first to demonstrate both symmetrical and asymmetrical cell divisions with CSDs in OSCC CSC. Further, the results suggest that CSCs may undergo altered behavior, including therapeutic resistance as a result of chromosomal instability due to chromosome segregation defects.

Abstract 1403: SOX2 as a marker of oral cancer stem-like cells: Cell division patterns and chromosomal instability

Introduction: Oral squamous cell carcinoma (OSCC) is a serious public health problem, caused primarily by smoking and alcohol consumption. The prognosis remains poor due in part to therapeutic resistance. The cancer stem cell (CSC) theory posits that oral cancer stem-like cells (CSLCs) show unique characteristics, including self-renewal by symmetric cell division and tumor recapitulation by asymmetric cell division. Identifying markers of CSLCs is an important step in better understanding the biology of these cells. Chromosomal instability is a feature of cancer cells that leads to altered gene copy numbers; some of which is caused by chromosome segregation defects (CSDs). We observed previously that chromosomal instability occurs in colon CSLCs. Here, we compare instability between OSCC CSLCs and non-CSLCs from the same tumors. Methods: We assessed CSLCs in two OSCC cell lines (UPCI:SCC040 and UPCI:SCC131) using spheroid enrichment assays. We examined the marker, SOX2 in our cell lines using double antibody staining with division and other stemness markers. We then evaluated the cell division patterns of CSLCs marked by SOX2 to test the hypothesis that cell division patterns change from asymmetric to symmetric after ionizing radiation (IR) treatment. Finally, we analyzed the CSDs after IR in CSLCs marked by SOX2 compared to non-CSLCs. Results: Both cell lines formed spheroids. Strongly SOX2-positive cells were a rare population (∼3%) in our OSCC cell lines. SOX2 positivity was highly correlated with other CSLC markers, including CD44, CD133 and BMI1. A cluster plot of the mitotic pair analysis revealed that SOX2-positive cells divide both symmetrically and asymmetrically (p Conclusions: Our results show that therapeutic intervention does not change the ratio of symmetric to asymmetric cell division in CSLCs. These results suggest that only therapies that target the self-renewal potential of CSLCs might effectively eradicate these cells. CSLCs showed less chromosomal instability as assessed by CSDs, possibly due either to the relative quiescence of these cells or enhanced checkpoints that result in cell death when CSDs are present. Cell division and chromosome segregation patterns in CSLCs may provide insight into the biology of therapeutic resistance in CSLCs and reveal strategies to eradicate these cells. Keywords: Oral squamous cell carcinoma (OSCC), Cancer stem-like cells (CSLCs), ionizing radiation (IR), chromosome segregation defects (CSDs). Citation Format: Hatem Kaseb, Dale Lewis, Sussane Gollin. SOX2 as a marker of oral cancer stem-like cells: Cell division patterns and chromosomal instability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1403. doi:10.1158/1538-7445.AM2015-1403

Abstract 4499: Loss of ATM (distal chromosome 11q) as a prognostic marker in HPV-negative head and neck squamous cell carcinomas

Introduction: HPV-negative head and neck squamous cell carcinomas (HNSCC) represent a subset of head and neck cancers with poor response to treatment and worse prognosis compared to HPV-positive HNSCC. In our prior pre-clinical experiments with HNSCC cell lines, we demonstrated that copy number loss of ATM as a marker for loss of distal chromosome 11q loss leads to defectiveγ-H2AX focus formation, increased chromosomal instability, and reduced sensitivity to ionizing radiation. We hypothesize that in HPV-negative HNSCC, ATM loss is a biomarker for poor outcome. Methods: We performed a single center, retrospective analysis of 42 HNSCC patients to determine if ATM loss is associated with poor overall survival in HPV-negative HNSCC. We performed fluorescence in situ hybridization using probes to cyclin D1 (CCND1) and ATM to assess copy number changes in paraffin sections from HNSCC tumors. Disease-specific survival was defined as time from the date of tissue procurement (surgery or biopsy) until death from disease (HNSCC). Patients who were alive at last follow-up or had died from causes unrelated to their disease were censored. Disease-specific survival by ATM loss status was estimated by the Kaplan-Meier method and tested for a difference by a two-tailed log rank test. Three covariates, age, T stage and N stage were investigated and the effect of ATM loss upon disease-specific survival was assessed with proportional hazards regression by adjusting for these covariates as needed to determine if ATM loss could be considered independently associated with cancer mortality. Results and Conclusions: HPV-negative HNSCC patients with ATM copy number loss have a median disease-specific survival of only 19 months compared to 65 months for patients without ATM loss (log rank p = .0401). We investigated whether the association between ATM loss and disease-specific survival was due to the confounding influence of other clinical variables. Age and T stage were modestly associated with survival, but neither covariate was correlated with ATM loss. We estimated the hazard ratio for ATM loss alone and conditional upon age and T stage was unchanged (.040 alone vs .047 adjusting for age and T stage). Therefore, distal 11q loss, marked by copy number loss of ATM appears to increase the risk of disease-specific mortality independently of age, N stage and T stage. Our results demonstrate that ATM loss in HPV-negative HNSCC may be prognostic of poor outcome and novel therapeutic strategies may be required in this subset of patients. Further studies to elucidate the mechanisms of distal 11q loss and study potential treatment options are underway in our laboratory. 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 4499. doi:1538-7445.AM2012-4499