Quiescent Tumor Research Articles

Abstract 332: Extracellular matrix stiffness regulates metabolic state in metastatic, but not quiescent, breast carcinoma cells

Abstract Increased breast density is associated with a 4-6 fold increased risk of developing breast cancer, and is associated with an increase in deposition of extracellular matrix (ECM) proteins, most abundantly collagen I. Collagen, like other ECM proteins, plays a structural role fundamental to tissue organization. High levels of collagen deposition correspond with a stiffer ECM, which is emerging as an important regulator of cell proliferation and tumor progression. Our previously reported microarray implicated changes in mammary epithelial cell metabolism in response to increased matrix stiffness, consistent with the expanding role of the ECM in tumor cell signaling. Here we report that increased matrix stiffness regulates the expression of pyruvate dehydrogenase kinase 1, a key regulator between lactic acid production and pyruvate entry into the mitochondria, in highly metastatic 4T1 breast carcinoma cells. Interestingly, we do not observe this same metabolic regulation in quiescent (dormant) 4T07 tumor cells of the same lineage. These alterations in protein expression correlated to changes in cellular NADH metabolism observed by several independent approaches, including metabolic flux analysis and quantitative imaging fluorescence lifetime microscopy. Thus, we find that alterations in collagen stiffness cause metabolic shifts between oxidative phosphorylation and aerobic glycolysis in highly metastatic cells, but not in quiescent cells. These findings identify stiffness of the ECM as an important regulator of metabolic state, and further identify quiescence as a dominant trait that is not overcome by ECM stiffness. Citation Format: Brett A. Morris, Brian Burkel, Suzanne Ponik, Kevin Eliceiri, Julio Aguirre-Ghiso, John Condeelis, James Castracane, Patricia J. Keely. Extracellular matrix stiffness regulates metabolic state in metastatic, but not quiescent, breast carcinoma cells. [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 332. doi:10.1158/1538-7445.AM2015-332

Mitotic quiescence, but not unique “stemness,” marks the phenotype of bone metastasis-initiating cells in prostate cancer

This study aimed to identify subpopulations of prostate cancer cells that are responsible for the initiation of bone metastases. Using rapidly dividing human prostate cancer cell lines, we identified mitotically quiescent subpopulations (<1%), which we compared with the rapidly dividing populations for patterns of gene expression and for their ability to migrate to the skeletons of athymic mice. The study used 2-photon microscopy to map the presence/distribution of fluorescently labeled, quiescent cells and luciferase expression to determine the presence of growing bone metastases. We showed that the mitotically quiescent cells were very significantly more tumorigenic in forming bone metastases than fast-growing cells (55 vs. 15%) and had a unique gene expression profile. The quiescent cells were not uniquely stem cell like, with no expression of CD133 but had the same level expression of other putative prostate stem cell markers (CD44 and integrins α2/β1), when compared to the rapidly proliferating population. In addition, mitotic quiescence was associated with very high levels of C-X-C chemokine receptor type 4 (CXCR4) production. Inhibition of CXCR4 activity altered the homing of quiescent tumor cells to bone. Our studies suggest that mitotic dormancy is a unique phenotype that facilitates tumor cell colonization of the skeleton in prostate cancer.

Trg-deficient Salmonella colonize quiescent tumor regions by exclusively penetrating or proliferating

Chemotherapeutics fail to effectively treat tumors because they cannot reach quiescent regions far from blood vessels. Motile Salmonella are an attractive delivery system that could break this therapeutic barrier. However, little is known about the dissemination and tissue penetration of individual bacteria in tumors after intravenous administration. We hypothesized that eliminating the Trg receptor would improve accumulation in tumor quiescence. To test this hypothesis, we deleted the trg gene from nonpathogenic Salmonella. To quantify individual bacterial behavior, we measured tissue penetration in a tumor-on-a-chip device and measured colony localization in mouse tumors using immunofluorescence. In tumors in vitro and in mice, trg− Salmonella penetrated farther into tissue than control bacteria. This difference in localization was caused by the inability to sense sugars in well perfused tissue. Three distinct bacterial phenotypes were observed: proliferating, penetrating, and inactive. Large proliferating colonies, containing more than 40% of individual bacteria, only formed less than 60μm from blood vessels. Small colonies, in comparison, were present both near (inactive) and far (penetrating) from vessels. The farthest was 361.2μm from a vessel, demonstrating the ability to target avascular regions. In addition, colonization was most pronounced in poorly vascularized tumor regions. We show that deletion of trg amplifies Salmonella accumulation in quiescent tumor regions, and, for the first time, identify biological processes that control bacterial distribution in tumors. Understanding how Salmonella penetrate tissue, target quiescence and specifically replicate in tumors are essential steps toward creating a tightly controlled, tunable bacterial therapy.

Stochastic model for tumor control probability: effects of cell cycle and (a)symmetric proliferation.

BackgroundEstimating the required dose in radiotherapy is of crucial importance since the administrated dose should be sufficient to eradicate the tumor and at the same time should inflict minimal damage on normal cells. The probability that a given dose and schedule of ionizing radiation eradicates all the tumor cells in a given tissue is called the tumor control probability (TCP), and is often used to compare various treatment strategies used in radiation therapy.MethodIn this paper, we aim to investigate the effects of including cell-cycle phase on the TCP by analyzing a stochastic model of a tumor comprised of actively dividing cells and quiescent cells with different radiation sensitivities. Moreover, we use a novel numerical approach based on the method of characteristics for partial differential equations, validated by the Gillespie algorithm, to compute the TCP as a function of time.ResultsWe derive an exact phase-diagram for the steady-state TCP of the model and show that at high, clinically-relevant doses of radiation, the distinction between active and quiescent tumor cells (i.e. accounting for cell-cycle effects) becomes of negligible importance in terms of its effect on the TCP curve. However, for very low doses of radiation, these proportions become significant determinants of the TCP. We also present the results of TCP as a function of time for different values of asymmetric division factor.ConclusionWe observe that our results differ from the results in the literature using similar existing models, even though similar parameters values are used, and the reasons for this are discussed.

The immune-related role of BRAF in melanoma

Background: The existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recently recognized in several types of cancer. The activation of a Th1 type of immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy. However, whether such dichotomy depends on the genetic make-up of individual cancers is not known yet. BRAF and NRAS mutations are commonly acquired during melanoma progression. Here we explored the role of BRAF and NRAS mutations in influencing the immune phenotype based on a classification previously identified by our group. MethodsOne-hundred-thirteen melanoma metastases underwent microarray analysis and BRAF and NRAS genotyping. Allele-specific PCR was also performed in order to exclude low-frequency mutations. ResultsComparison between BRAF and NRAS mutant versus wild type samples identified mostly constituents or regulators of MAPK and related pathways. When testing gene lists discriminative of BRAF, NRAS and MAPK alterations, we found that 112 BRAF-specific transcripts were able to distinguish the two immune-related phenotypes already described in melanoma, with the poor phenotype associated mostly with BRAF mutation. Noteworthy, such association was stronger in samples displaying low BRAF mRNA expression. However, when testing NRAS mutations, we were not able to find the same association. ConclusionThis study suggests that BRAF mutation-related specific transcripts associate with a poor phenotype in melanoma and provide a nest for further investigation.

Cancer immunotherapy: treatment of established breast cancer or prevention of tumor relapse and metastasis? (TUM2P.903)

Abstract Cancer vaccines and adoptive immunotherapies (AITs) are being tested both in preventive and therapeutic settings. In order to determine the most effective immunotherapeutic strategy, we investigated the efficacy of AIT against established advanced carcinoma and minimal residual disease. We used Decitabine to improve immunogenicity of the tumor cells by inducing the expression of cancer testis antigens (CTA), in vivo. Tumor-sensitized immune cells were activated and reprogrammed, ex vivo, using bryostatin 1 and ionomycin, along with IL-2, IL-7, and IL-15; this produced a central memory phenotype in 73% of T cells, and an activated phenotype in 27% of NK cells and 94% of NKT cells. FVBN202 transgenic mice were protected from developing lung metastases following AIT, but failed to cure established lung metastases. The reprogrammed immune cells also demonstrated immunological memory that protected animals from recall tumor challenge. In vitro studies showed that reprogrammed immune cells were able to kill 82% of viable Adriamycin-treated quiescent tumor cells. Our results suggest that AIT could be efficacious against chemotherapy-resistant dormant tumor cells in a setting of low tumor burden and prevent distant metastasis of breast cancer. These findings are significant because breast cancer mortality results from metastatic disease that develops from residual tumor cells remaining dormant, sometimes for prolonged periods following conventional therapies.

Therapeutic Properties of Aerobic Training After a Cancer Diagnosis: More Than a One-Trick Pony?

Until recently there was a prevailing dogma that cancer patients may be unable to safely participate in, benefit from, or tolerate structured exercise training/rehabilitation. Nevertheless, two factors pointed to the potential benefit of exercise in cancer patients: 1) the robust efficacy of exercise to favorably impact multiple physiological and psychosocial outcomes in noncancer clinical populations with similar symptomatology and limitations to exercise and 2) the emergence and importance of cancer survivorship. These factors provided the ideal platform and rationale to launch initial studies testing the safety, tolerability, and efficacy of exercise in patients with cancer (1). The past decade has witnessed a relative explosion in research, as well as clinical interest, in the application of exercise, as well as more general physical activity, in the context of cancer control efforts (1).

The dependency of compound biological effectiveness factors on the type and the concentration of administered neutron capture agents in boron neutron capture therapy.

PurposeTo examine the effect of the type and the concentration of neutron capture agents on the values of compound biological effectiveness (CBE) in boron neutron capture therapy.Methods and materialsAfter the subcutaneous administration of a 10B-carrier, boronophenylalanine-10B (BPA) or sodium mercaptododecaborate-10B (BSH), at 3 separate concentrations, the 10B concentrations in tumors were measured by γ-ray spectrometry. SCC VII tumor-bearing C3H/He mice received 5-bromo-2′-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells, then treated with BPA or BSH. Immediately after reactor neutron beam irradiation, during which intratumor 10B concentrations were kept at levels similar to each other, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of BrdU-unlabeled quiescent (Q) and total (= P + Q) tumor cells were assessed based on the frequencies of micronucleation using immunofluorescence staining for BrdU.ResultsThe CBE values were higher in Q cells and in the use of BPA than total cells and BSH, respectively. In addition, the higher the administered concentrations were, the smaller the CBE values became, with a clearer tendency in the use of BPA than BSH. The values for neutron capture agents that deliver into solid tumors more dependently on uptake capacity of tumor cells became more changeable.ConclusionTumor characteristics, such as micro-environmental heterogeneity, stochastic genetic or epigenetic changes, or hierarchical organization of tumor cells, are thought to partially influence on the value of CBE, meaning that the CBE value itself may be one of the indices showing the degree of tumor heterogeneity.

Hybrid discrete-continuum model of tumor growth considering capillary points

A hybrid discrete-continuum model of tumor growth in the avascular phase considering capillary points is established. The influence of the position of capillary points on tumor growth is also studied by simulation. The results of the dynamic tumor growth and the distribution of oxygen, matrix-degrading enzymes, and extracellular matrixconcentration in the microenvironment with respect to time are shown by graphs. The relationships between different oxygenated environments and the numbers of surviving, dead, proliferative, and quiescent tumor cells are also investigated.

Quantitative receptor-based imaging of tumor proliferation with the sigma-2 ligand [(18)F]ISO-1.

The sigma-2 receptor is expressed in higher density in proliferating (P) tumor cells versus quiescent (Q) tumor cells, thus providing an attractive target for imaging the proliferative status (i.e., P:Q ratio) of solid tumors. Here we evaluate the utility of the sigma-2 receptor ligand 2-(2-[18F]fluoroethoxy)-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl)-5-methyl-benzamide, [18F]ISO-1, in two different rodent models of breast cancer. In the first study, small animal Positron Emission Tomography (PET) imaging studies were conducted with [18F]ISO-1 and 18FDG in xenografts of mouse mammary tumor 66 and tracer uptake was correlated with the in vivo P:Q ratio determined by flow cytometric measures of BrdU-labeled tumor cells. The second model utilized a chemically-induced (N-methyl-N-nitrosourea [MNU]) model of rat mammary carcinoma to correlate measures of [18F]ISO-1 and FDG uptake with MR-based volumetric measures of tumor growth. In addition, [18F]ISO-1 and FDG were used to assess the response of MNU-induced tumors to bexarotene and Vorozole therapy. In the mouse mammary 66 tumors, a strong linear correlation was observed between the [18F]ISO-1 tumor: background ratio and the proliferative status (P:Q ratio) of the tumor (R = 0.87). Similarly, measures of [18F]ISO-1 uptake in MNU-induced tumors significantly correlated (R = 0.68, P<0.003) with changes in tumor volume between consecutive MR imaging sessions. Our data suggest that PET studies of [18F]ISO-1 provide a measure of both the proliferative status and tumor growth rate, which would be valuable in designing an appropriate treatment strategy.

Hybrid discrete-continuum model of tumor growth considering capillary points

A hybrid discrete-continuum model of tumor growth in the avascular phase considering capillary points is established. The influence of the position of capillary points on tumor growth is also studied by simulation. The results of the dynamic tumor growth and the distribution of oxygen, matrix-degrading enzymes, and extracellular matrix-concentration in the microenvironment with respect to time are shown by graphs. The relationships between different oxygenated environments and the numbers of surviving, dead, proliferative, and quiescent tumor cells are also investigated.

Chaperone-mediated autophagy degrades mutant p53

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells.

Activated Pancreatic Stellate Cells Sequester CD8+ T Cells to Reduce Their Infiltration of the Juxtatumoral Compartment of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. We used tissue microarray analysis to compare immune cell infiltration of different pancreaticobiliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis) and juxtatumoral stromal (<100 μm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We also analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice and the effects of all-trans retinoic acid (ATRA) on these processes. Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase(+) and CD68(+) cells compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8(+), FoxP3(+), CD56(+), or CD20(+) cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8(+) T cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8(+) T cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. Invitro migration assays showed that CD8(+) T cells, from patients with PDAC, had increased chemotaxis toward activated PSCs, which secrete CXCL12, compared with quiescent PSCs or tumor cells. These effects could be reversed byknockdown of CXCL12 or treatment of PSCs with ATRA. Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8(+) T cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective antitumor immune response.

The σ2 Receptor: A Novel Protein for the Imaging and Treatment of Cancer

The σ2 receptor is an important target for the development of molecular probes in oncology because of its 10-fold higher density in proliferating tumor cells compared with that in quiescent tumor cells and because of the observation that σ2 receptor agonists are able to kill tumor cells via apoptotic and nonapoptotic mechanisms. Although recent evidence indicates that the σ2 receptor binding site is localized within the progesterone receptor membrane component 1 (PGRMC1), most information regarding this protein has been obtained using either radiolabeled or fluorescent receptor-based probes and from biochemical analysis of the effect of σ2 selective ligands on cells grown in culture. This article reviews the development of σ2 receptor ligands and presents an overview of how they have been used in vitro and in vivo to increase our understanding of the role of the σ2 receptor in cancer and proliferation.

Abstract 2164: Mirk/dyrk1B kinase inhibitor reduces the viability of patient-derived ovarian cancer ascites cells and the size of murine tumor xenografts.

Abstract Ovarian cancer is one of the most deadly malignancies for women because it typically presents at a late stage when conventional therapies have limited effect. Ovarian cancer often metastasizes by forming peritoneal ascites, consisting of tumor spheroids within a large fluid volume. Others have shown that ascites tumor spheroids are largely quiescent with about 85% in G0/G1, but retain the capacity to re-enter the cell cycle after attachment to the peritoneal cavity walls. Mirk/dyrk1B kinase is most highly expressed when cancer cells are quiescent, and is known to maintain cells in quiescence by destabilizing cyclin D isoforms and by phosphorylating a member of the DREAM complex that enables p130/Rb2 to sequester transcription factors. In earlier studies, Mirk/dyrk1B kinase inhibition or depletion in quiescent adherent cancer cells, either ovarian or pancreatic, allowed cells to escape quiescence, enter cycle prematurely and suffer DNA damage and subsequent apoptosis. No such effects were seen in normal diploid cells in which Mirk/dyrk1B kinase was in low abundance and had low activity, and diploid cells have a normal complement of CDK inhibitors to prevent escape from quiescence. We now show that spheroids from three ovarian cancer cell lines and the Panc1 pancreatic cancer cell line are largely quiescent, and enriched in Mirk/dyrk1B kinase, the quiescence protein p130/Rb2 and the CDK inhibitor p27. Treatment with two novel Mirk/dyrk1B kinase inhibitors led to a decrease in spheroid quiescence markers, and up to a 7-fold decrease in spheroid volume and viable cell numbers. Significantly, Mirk is expressed in patient-derived ovarian cancer ascites spheroids, and ascites treatment with the most potent Mirk/dyrk1B kinase inhibitor led first to an induction of apoptosis markers, then to a disruption of spheroid structure, and finally to a 90% loss in viable tumor cells. This inhibitor, at concentrations that killed patient-derived ovarian cancer ascites spheroid cells in vitro, had no detectable toxicity in mice, not decreasing mouse weight, but reduced the size of Panc1 xenografts up to 3-fold, while increasing the percent of Ki67 positive tumor cells. Both observations were consistent with the model that Mirk/dyrk1B kinase inhibition allowed quiescent tumor cells to enter cycle prematurely where they would die. Citation Format: Xiaobing Deng, Jing Hu, Mary Cunningham, Bertrand Leblond, Thierry Besson, Anne-Sophie Casagrande, Laurent Désiré, Eileen A. Friedman. Mirk/dyrk1B kinase inhibitor reduces the viability of patient-derived ovarian cancer ascites cells and the size of murine tumor xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2164. doi:10.1158/1538-7445.AM2013-2164

Effect of Tirapazamine and Mild Temperature Hyperthermia on the Recovery from Radiation-Induced Damage in Pimonidazole-Unlabeled Quiescent Tumor Cell Population

The aim in this study is to examine the effect of tirapazamine (TPZ) and mild temperature hyperthermia (MTH) on the repair of radiation-induced damage in pimonidazole-unlabeled quiescent (Q) tumor cells. Labeling of proliferating (P) cells in C57BL/6J mice bearing EL4 tumors was achieved by continuous administration of 5-bromo-2-deoxyuridine (BrdU). Tumors were irradiated with γ-rays at 1 h after the administration of pimonidazole followed by TPZ treatment or MTH. Twenty-four hours later, assessment of the responses of Q and total (= P + Q) cells were based on the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of the pimonidazole-unlabeled tumor cell fractions was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole. With γ-rays only, the pimonidazole-unlabeled cell fraction showed significantly enhanced radio-sensitivity compared with the whole cell fraction more remarkably in Q cells than total cells. However, a significantly greater decrease in radio-sensitivity in the pimonidazole-unlabeled than the whole cell fraction, evaluated using a delayed assay, was more clearly observed in Q cells than total cells. Post-irradiation MTH more remarkably repressed the decrease in radio-sensitivity in the Q cell than the total cells. Post-irradiation TPZ administration produced a large radio-sensitizing effect on both total and Q cells, especially on Q cells. On the other hand, in pimonidazole-unlabeled cell fractions in both total and Q cells, TPZ suppressed the reduction in sensitivity due to delayed assay much more efficiently than MTH, whereas no radio-sensitizing effect was produced. Not only through suppressing the recovery from radiation-induced damage but also through radio-sensitizing effect, post-irradiation TPZ administration is very useful for repressing the increase in the difference in radio-sensitivity due to the delayed assay not only between total and Q tumor cells but also between the pimonidazole-unlabeled and the whole cell fractions within the total and Q tumor cells.

Wortmannin efficiently suppresses the recovery from radiation-induced damage in pimonidazole-unlabeled quiescent tumor cell population

Labeling of proliferating (P) cells in mice bearing EL4 tumors was achieved by continuous administration of 5-bromo-2′-deoxyuridine (BrdU). Tumors were irradiated with γ-rays at 1 h after pimonidazole administration followed by caffeine or wortmannin treatment. Twenty-four hours later, assessment of the responses of quiescent (Q) and total (= P + Q) cell populations were based on the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of the pimonidazole-unlabeled tumor cell fractions was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole. The pimonidazole-unlabeled cell fraction showed significantly enhanced radio-sensitivity compared with the whole cell fraction more remarkably in Q cells than total cells. However, a significantly greater decrease in radio-sensitivity in the pimonidazole-unlabeled than the whole cell fraction, evaluated using an assay performed 24 hours after irradiation, was more clearly observed in Q cells than total cells. In both the pimonidazole-unlabeled and the whole cell fractions, wortmannin efficiently suppressed the reduction in sensitivity due to delayed assay. Wortmannin combined with γ-ray irradiation is useful for suppressing the recovery from radiation-induced damage especially in the pimonidazole-unlabeled cell fraction within the total and Q tumor cell populations.

A Tumor Growth Inhibition Model for Low-Grade Glioma Treated with Chemotherapy or Radiotherapy

To develop a tumor growth inhibition model for adult diffuse low-grade gliomas (LGG) able to describe tumor size evolution in patients treated with chemotherapy or radiotherapy. Using longitudinal mean tumor diameter (MTD) data from 21 patients treated with first-line procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and vincristine (PCV) chemotherapy, we formulated a model consisting of a system of differential equations, incorporating tumor-specific and treatment-related parameters that reflect the response of proliferative and quiescent tumor tissue to treatment. The model was then applied to the analysis of longitudinal tumor size data in 24 patients treated with first-line temozolomide (TMZ) chemotherapy and in 25 patients treated with first-line radiotherapy. The model successfully described the MTD dynamics of LGG before, during, and after PCV chemotherapy. Using the same model structure, we were also able to successfully describe the MTD dynamics in LGG patients treated with TMZ chemotherapy or radiotherapy. Tumor-specific parameters were found to be consistent across the three treatment modalities. The model is robust to sensitivity analysis, and preliminary results suggest that it can predict treatment response on the basis of pretreatment tumor size data. Using MTD data, we propose a tumor growth inhibition model able to describe LGG tumor size evolution in patients treated with chemotherapy or radiotherapy. In the future, this model might be used to predict treatment efficacy in LGG patients and could constitute a rational tool to conceive more effective chemotherapy schedules.

Abstract 2858: Mirk/dyrk1B kinase inhibition increases ROS and apoptosis in ovarian cancer cells, but has little effect on normal diploid cells

Abstract Quiescent cancer cells in vivo, including stem cell populations, are relatively insensitive to most chemotherapeutic drugs and radiation. Tumor cells in culture can be placed in G0 by serum or nutrient deprivation, and then can be targeted by a small molecule Mirk kinase inhibitor. Mirk/dyrk1B levels are elevated up to 10-fold in quiescent tumor cells where Mirk blocks cell cycling and increases viability by decreasing reactive oxygen species (ROS). In prior studies Mirk was detected in 75% of ovarian cancers, and was amplified in a subset. In contrast, Mirk expression was quite low in most normal cell types and Mirk knockout or depletion had no detectable effects on normal cells. Response to a Mirk kinase inhibitor was compared between two strains of normal diploid fibroblasts, early passage cultures of normal human ovarian epithelial cells, and a series of ovarian cancer cell lines where Mirk had been shown to be an active kinase. Serum-starved normal diploid cells entered a G0 quiescent state and had minimal response to the Mirk inhibitor, but serum-starved cancer cells were kept in cycle by the Mirk inhibitor, which reduced E2F4 sequestration by p130/Rb2 by 2-3fold. Mirk inhibitor treated cancer cultures had several-fold more sub-G0/G1 apoptotic cells and about 30-fold higher levels of the apoptotic proteins cleaved PARP and cleaved caspase 3 compared with normal diploid cells. The Mirk inhibitor increased total ROS levels and superoxide levels only in the cancer cells, in a rapid time-course of 2-3 days. Cancer cell loss after Mirk kinase inhibitor treatment was due at least in part to ROS, since the ROS scavenger N-acetyl cysteine reduced the amount of cleaved PARP and the extent of cancer cell loss. The Mirk inhibitor decreased the EC50 of cisplatin up to 9-fold in ovarian cancer cells. Thus ovarian cancer cells, which exhibit elevated Mirk expression/amplification and active Mirk kinase, were shown to be more susceptible to the Mirk kinase inhibitor than normal diploid cells. 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 2858. doi:1538-7445.AM2012-2858

Histone Deacetylase 6 (HDAC6) Deacetylates Survivin for Its Nuclear Export in Breast Cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.