Citations

Abstract Abstract 29 Acute lymphoblastic leukemia (ALL) cells are frequently characterized by abnormal numbers of chromosomes, a condition known as aneuploidy. Aneuploidy in ALL is not just a curiosity, but impacts on the prognosis and risk stratification in children. Additionally, aneuploidy itself contributes to the development of cancer by dysregulating gene expression, and driving the loss of heterozygosity of mutated tumor suppressor genes. The molecular underpinnings of aneuploidy in ALL are largely unknown. The mitotic checkpoint is a complex pathway that acts in mitosis to prevent the premature onset of anaphase that leads to improper segregation of chromosomes and aneuploidy. Through the concerted action of many intermediates, the mitotic checkpoint functions to inhibit the activity of a large ubiquitin E3 ligase complex known as the anaphase promoting complex/cyclosome (APC/C) that drives the separation of paired sister chromatids by effecting the ubiquitin-dependent degradation of cyclin B1 and securin. The de-ubiquitinating enzyme USP44 was recently identified as an important regulator of mitotic progression that assists in maintaining mitotic checkpoint signaling until all chromosomes are properly attached to the mitotic spindle. Microarray studies of childhood acute leukemias have observed high levels of USP44 in T-cell ALL, but the significance of this observation is unclear. Here we show that USP44 is indeed over-expressed in T-ALL in children and that its presence may contribute to aneuploidy that is frequently observed in this disease. We compared the expression of USP44 in a series of 24 samples from patients with T-ALL to that found in ten samples of peripheral T-cells isolated from healthy donors, using quantitative real-time PCR. There was a dramatic increase in the expression of USP44 in T-ALL with 18 out of 24 cases having at least 3-fold increase, with an average of 16-fold increase, in USP44 mRNA. We next studied the consequences of USP44 over-expression in primary cells using live-cell microscopy. We find that excess USP44 leads to dramatic and significant increase in chromosome missegregation with most defects involving lagging chromosomes due to improper chromosome attachment to the mitotic spindle. We observe that these mitotic errors lead to the development of aneuploidy, through a mechanism that requires USP44 catalytic activity. These effects are accompanied by reinforced mitotic checkpoint signaling that leads to delayed progression through mitosis, and a prolonged duration of mitotic arrest in cells exposed to spindle poisons such as nocodazole or paclitaxel. At the molecular level, we observe that excess USP44 leads to enhanced binding of the mitotic checkpoint effector Mad2 to the APC/C and increased levels of cyclin B1 in early mitosis. Taken together, we conclude that USP44 is a chromosome instability gene that is frequently expressed at high levels in T-ALL cells, and that this may contribute to the pathogenesis of T-ALL by leading to chromosome shuffling and aneuploidy. These data have important implications in our understanding of the pathogenesis of this disease and may contribute to understanding the therapeutic effectiveness of novel aurora inhibitors in cancer. Disclosures: No relevant conflicts of interest to declare.

6520 Background: Breast cancer follow-up guidelines recommend imaging for distant metastases only in the presence of signs/symptoms. However, data supporting this recommendation predates the current era of improved imaging and targeted therapies based on molecular subtype. The objective was to assess the relationship between mode of distant recurrence detection and survival. Methods: A stage-stratified random sample of Stage II-III breast cancer patients diagnosed in 2006-7 was selected from NCDB records from 1,217 CoC-accredited facilities (10/hospital n = 10,853). Women were categorized by subtype: 1) ER or PR+/HER2-; 2) ER and PR-/Her2- (triple negative); 3) HER2+. Medical records abstracted for 5-years post-surgery supplemented NCDB data and assessed distant recurrence and mode of detection (prompted by signs/symptoms or surveillance imaging), imaging (chest CT, abdomen/pelvis CT/MRI, head CT/MRI, bone scan, PET/CT), death date. The relationship between mode of recurrence detection and days from initial cancer diagnosis to death was assessed using propensity-weighted multivariable Cox proportional hazards regression stratified by subtype. Propensity weights, based on receipt of surveillance systemic imaging, accounted for sociodemographic and tumor/treatment factors. Results: 5-year distant recurrence was 22.3% for triple negative, 14.8% HER2+, and 11.2% for ER or PR+/ HER2- patients. Asymptomatic imaging detected recurrence in 22.9% and signs/symptoms in 77.1%. Patients with asymptomatic as compared to sign/symptom detected recurrences had reduced risk of death in 5 years if triple negative (HR = 0.68, 95% CI = 0.50-0.93) or HER2+ (HR = 0.40, 95% CI = 0.24-0.65) with no significant association for ER or PR+/HER2- (HR = 1.2, 95% CI = 0.88-1.51). This translated to a between-group difference in weighted median survival of 5 months for triple negative and 13 months for HER2+ patients. Conclusions: This is the first nationally representative study to show a survival advantage with asymptomatic detection of distant metastases for patients, with the benefit limited to triple negative and HER2+ disease. Further research to confirm observational findings is warranted.

Abstract Tumor-associated macrophages (TAM) play a critical role in cancer development and progression. However, the heterogeneity of TAM presents a major challenge to identify clinically relevant markers for protumor TAM. Here, we report that expression of adipocyte/macrophage fatty acid–binding protein (A-FABP) in TAM promotes breast cancer progression. Although upregulation of A-FABP was inversely associated with breast cancer survival, deficiency of A-FABP significantly reduced mammary tumor growth and metastasis. Furthermore, the protumor effect of A-FABP was mediated by TAM, in particular, in a subset of TAM with a CD11b+F4/80+MHCII−Ly6C− phenotype. A-FABP expression in TAM facilitated protumor IL6/STAT3 signaling through regulation of the NFκB/miR-29b pathway. Collectively, our results suggest A-FABP as a new functional marker for protumor TAM. Significance: These findings identify A-FABP as a functional marker for protumor macrophages, thus offering a new target for tumor immunotherapy. Cancer Res; 78(9); 2343–55. ©2018 AACR.

Abstract Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. Two common alterations in GBM are loss of the tumor suppressor PTEN and upregulation of the oncogenic receptor tyrosine kinase MET. We uncovered a new connection between PTEN and MET expressions and functions in GBM. We found that PTEN expression in GBM cell lines (U87, U373, A172, LNZ-308) and primary cells (GBM8) inhibits MET expression. We uncovered the molecular basis and functional significance of this connection. Based on our previous work and published literature, PTEN stabilizes p53, p53 induces miR-34a expression, and miR-34a inhibits MET expression. We therefore hypothesized that PTEN inhibits MET by regulating p53 and miR-34a and that the PTEN tumor suppressive effects are partly mediated by MET downregulation. To verify the hypothesis, we first showed that PTEN restoration leads to miR-34a upregulation in GBM cells. We then showed that this latter effect is p53-dependent as p53 knockdown with a specific shRNA partly reversed PTEN induction of miR-34a. We also showed that PTEN-induced downregulation of MET expression can be rescued by inhibition of miR-34a with an anti-miR3a. We therefore established a new molecular cascade that links PTEN, p53, miR-34a and MET in GBM. To determine the functional relevance of this cascade, we assessed the effects of PTEN on GBM cell death and invasion also in the settings of shRNA-mediated knockdown of p53 and anti-miR-mediated inhibition of miR-34a. PTEN strongly induced GBM cell death (measured by Annexin V/7AAD flow cytometry) and inhibited transwell invasion though a collagen IV matrix. The PTEN effects on cell death and invasion were partly reduced by p53 knock-down and miR-34a inhibition. This demonstrates that the tumor suppressive effects of PTEN in GBM are partly mediated by the PTEN/p53/miR-34a/MET cascade. In ongoing experiments, we are investigating the expressions of PTEN, MET, p53 and miR-34a in human GBM tissues to determine if there are potential correlations that are consistent with our above findings. Altogether, this study establishes a new molecular and functional link between PTEN and MET that involves p53 and miR-34a in GBM. Citation Format: Fadilla Guessous, Nichola A. Cruickshanks, Ying Zhang, David Schiff, Jann Sarkaria, Roger Abounader. PTEN tumor suppressive effects in glioblastoma are partly mediated by MET downregulation via a p53-miR-34a axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 345. doi:10.1158/1538-7445.AM2017-345

AbstractThe schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3‐deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X‐receptor (LXR) activation. The effects of 3dSB on ATP‐binding cassette transporter 1 levels and other LXR targets are similar to that of 25‐hydroxycholesterol, an LXR agonist. Unlike 25‐hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or β. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.

Rac1b mediates the matrix metalloproteinase–induced epithelial-mesenchymal transition in lung adenocarcinoma and is a potential therapeutic target in early-stage lung cancer.

Abstract During development, epithelial cells acquire mesenchymal characteristics through a process known as epithelial-mesenchymal transition (EMT). Substantial evidence now exists that EMT-related processes play a major role in tumor progression. EMT is a focus of interest in lung cancer as loss of E-cadherin expression confers increased resistance to several types of chemotherapy. In previous studies, we found that matrix metalloproteinase-3 (MMP3) induced EMT in mouse mammary epithelial cells through induction of Rac1b, an alternatively spliced isoform of Rac1. We now show that exposure to MMPs stimulates Rac1b and induction of EMT markers in human lung cancer cell lines, and that knockdown of Rac1b blocks MMP induced EMT. We also show that Rac1b expression levels in human lung tumor biopsies is predictive of patient outcome, and that Rac1b and MMP expression levels are correlated in these samples, suggesting a direct relationship. We also describe the generation of new transgenic mouse models for which MMP3 or Rac1b is inducibly expressed in lung epithelial cells; activation of kRAS in these mice reveals that expression of MMP3 or Rac1b promotes increased tumor number, size, and progression to adenocarcinoma as well as increased expression of EMT markers vimentin, sm-actin, col1a1, and Snail. We also show that expression of Rac1b in lung epithelial cells leads to spontaneous adenocarcinoma formation. These results demonstrate a direct linkage between induction of EMT and tumor progression, implicate Rac1b as a key mediator of these processes, and demonstrate that MMPs can directly stimulate all of these events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2318.