Cancer Stem Cell Fate Research Articles

Abstract LB-028: Mechanically tunable 3D microenvironments modulate tumor cell phenotype: Models of mechanotransduction and drug resistance in osteosarcoma

Abstract The past few decades have seen marked improvements in survival rates of osteosarcoma due to the advent of modern chemotherapy, radiotherapy, and surgical techniques. However, for patients presenting with metastatic disease, five-year survival rates have remained dismal, hovering around 20%. Repeated failures of clinical trials to confirm potential therapeutic options highlight the need for more accurate pre-clinical testing. Currently, the field of cancer research relies heavily on monolayer culture methods on hard plastic or glass in vitro models; there is a dearth of pre-clinical models that accurately recapitulate the tumor-microenvironment interactions. Matrix stiffness has been implicated in modulating intracellular signaling pathways that promote cancer cell survival, proliferation, and stem cell fate. We have developed a novel three-dimensional (3D) tumor model with variable mechanical properties in order to determine the effect of substrate stiffness and tissue architecture on osteosarcoma cell phenotype, plasticity, and response to therapy. We employed coaxial electrospinning techniques to fabricate highly porous fibrous mesh scaffolds that mimic the bone microenvironment. By controlling the ratio of poly(ϵ-caprolactone) (PCL) and gelatin (PCL:gelatin, core:shell, respectively) in constituent fibers, we were able to manipulate the range of tensile moduli of individual fibers over three orders of magnitude, from 68.91 ± 8.77 kPa to 66.05 ± 7.61 MPa. Osteosarcoma cells cultured in these variable mechanical environments responded by modulating the localization and expression of Hippo pathway regulators. YAP downregulation correlated with decreasing fiber stiffness while both YAP and TAZ had decreasing nuclear:cytoplasmic ratio in less stiff environments. Furthermore, the IGF-1/mTOR axis was downregulated in 3D conditions compared to monolayers and a strong upregulation of Sox2, a stem cell transcription factor, was observed in all 3D conditions. Correspondingly, in the presence of agents targeting the IGF-1/mTOR axis, dose response curves to doxorubicin indicated that IC50 values increase with decreasing substrate stiffness. These phenotypic changes indicate that osteosarcoma cells respond to both stiffness and architecture by modulating the Hippo and IGF-1R/mTOR pathways and increasing cancer stem cell qualities and chemoresistance. We sought to validate our model using osteosarcoma patient biopsies. Analysis of tumor samples from 36 osteosarcoma patients confirmed that YAP/TAZ localization and nuclear pIGF-1R/IGF-1R in our 3D models recapitulated phenotypes observed in patient samples. Our models highlight the need for incorporation of mechanical and architectural cues in the preclinical study of cancer biology as these signals have drastic impacts on osteosarcoma phenotypes and responses to therapy. Citation Format: Eric R. Molina, Letitia K. Chim, Maria C. Salazar, Shail M. Mehta, Brian A. Menegaz, Salah-Eddine Cherradi-Lamhamedi, Tejus Satish, David McCall, Sana Mohiuddin, Ana Maria Zaska, Katherine Jane Grande-Allen, Joseph A. Ludwig, Antonios G. Mikos. Mechanically tunable 3D microenvironments modulate tumor cell phenotype: Models of mechanotransduction and drug resistance in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-028.

Cancer stem cell fate determination: a nuclear phenomenon

Cancer stem cells (CSCs) are a subset of cells within the tumor bulk, with a potential to undergo self-renewal, differentiation, proliferation and metastasis. Cumulative evidences suggest that mainly CSCs are responsible for tumor recurrences and resistance towards chemo- and radiotherapy. Unlike non-stem cancer cells, CSCs can undergo self-renewal and differentiation through asymmetric cell division (ACD) similar to normal stem cells. Asymmetric division results in two daughter cells: a structural and functional copy of the mother cell (CSC) and another one destined to undergo differentiation, non-cancer stem cell. Apart from the involvement of various polarity-determining factors, transcription factors and epigenetic regulators, micro-RNAs also play a crucial role in cell-fate decision making. Eventually, two daughter cells with distinct fates are produced with an unequal inheritance of differentiation-linked fate determinants, centrosomes and other stemness associated factors. Violation of the mechanisms leading to ACD will compromise the generation of both new CSCs and progenitor cells within the tumor. Thus, manipulation of asymmetric division of CSCs may introduce new strategies for dual targeting of CSCs and the tumor bulk. In this review, we discuss how CSCs play a critical role in almost all the events of tumorigenesis. Apart from that, we also delineate how these CSCs exploit the phenomenon of ACD during cancer relapses or tumor recurrences to regenerate the entire tumor. Therefore, in a nutshell this review highlights the therapeutic relevance of CSCs for successful elimination of cancer and suggests a novel improvisation in CSC-targeting by perturbing its ACD mechanisms.

The SCRIB Paralog LANO/LRRC1 Regulates Breast Cancer Stem Cell Fate through WNT/β-Catenin Signaling.

SummaryTumor initiation, progression, and therapeutic resistance have been proposed to originate from a subset of tumor cells, cancer stem cells (CSCs). However, the current understanding of the mechanisms involved in their self-renewal and tumor initiation capacity remains limited. Here, we report that expression of LANO/LRRC1, the vertebrate paralog of SCRIB tumor suppressor, is associated with a stem cell signature in normal and tumoral mammary epithelia. Through in vitro and in vivo experiments including a Lano/Lrrc1 knockout mouse model, we demonstrate its involvement in the regulation of breast CSC (bCSC) fate. Mechanistically, we demonstrate that Lano/LRRC1-depleted cells secrete increased levels of WNT ligands, which act in a paracrine manner to positively deregulate the WNT/β-catenin pathway in bCSCs. In addition to describing the first function of LANO/LRRC1, our results suggest that its expression level could be used as a biomarker to stratify breast cancer patients who could benefit from WNT/β-catenin signaling inhibitors.

Abstract 2490: Characterization of the role of DPPA3 in stemness regulation in hepatocellular carcinoma by DNA methylation

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with a very poor prognosis, which is closely associated with cancer stem cell (CSC). Global RNA-sequencing was used in different developmental stages of hepatocytes, including embryonic stem cell, endoderm, liver progenitor, and premature hepatocyte, as well as two pairs of nontumor and tumor tissues from HCC patients. We found that Developmental Pluripotency Associated 3 (DPPA3) was highest expressed in the stage of liver progenitor cells, and was down-regulated along with development. In HCC patients, DPPA3 was expressed in less than 1% tumor cells, while no staining was detected in normal counterparts. Functional assays revealed that DPPA3 overexpression increased HCC cells proliferation rate, the ability of foci formation and colony formation in soft agar, as well as migration and invasion. Furthermore, DPPA3 significantly increased HCC cells sphere formation frequency and sensitivity to chemotherapeutic agent including cisplatin, 5-Fu, and Sorafenib. In order to investigate the role of DPPA3 in CSC maintenance and stemness regulation in HCC, whole genome methylation sequencing was applied to compare methylation status between DPPA3- and empty vector-transfected cells. Results showed that DPPA3 reduced CG methylation level within functional regions in the genome. Of the 541 differentially methylated regions, the majority of them were involved in developmental process and cell differentiation. Overall, our findings give a novel insight into how methylation regulation controls the fate of cancer stem cells. Citation Format: Qian Yan, Yu Zhang, Dandan Yu, Ngar Woon Kam, Xin-Yuan Guan. Characterization of the role of DPPA3 in stemness regulation in hepatocellular carcinoma by DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2490.

The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells

BackgroundTumor hypoxia is an independent prognostic factor associated with poor patient survival. Emerging evidence suggests that hypoxia can potentially maintain or enhance the stem cell phenotype of both normal stem cells and cancer cells. However, it remains to be determined whether cell fate is regulated in vivo by the hypoxic tumor microenvironment (TME).MethodsWe established a hypoxia-sensing xenograft model to identify hypoxic tumor cell in vivo primarily using human breast cancer cell lines MDA-MB-231 and MCF7. Hypoxic tumor cells were identified in situ by fluorescence of green fluorescence protein. They were further isolated from xenografts, purified and sorted by flow cytometry for detailed analysis of their stem cell characteristics.ResultsWe have found that hypoxic tumor cells freshly isolated from xenografts contain increased subpopulations of tumor cells with cancer stem cell (CSC)-like characteristics. The CSC characteristics of the hypoxic tumor cells are further enhanced upon re-implantation in vivo, whereas secondary xenografts derived from the non-hypoxic tumor cells remain similar to the primary xenografts. Interestingly, the phenotypes exhibited by the hypoxic tumor cells are stable and remain distinctively different from those of the non-hypoxic tumor cells isolated from the same tumor mass even when they are maintained under the same ambient culture conditions. Mechanistically, the PI3K/AKT pathway is strongly potentiated in the hypoxic tumor cells and is required to maintain the CSC-like phenotype. Importantly, the differential cell fates between hypoxic and non-hypoxic tumor cells are only found in tumor cells isolated from the hypoxic TME in vivo and are not seen in tumor cells treated by hypoxia in vitro alone.ConclusionsThese previously unknown observations suggest that the hypoxic TME may promote malignant progression and therapy resistance by coordinating induction, selection and/or preferential maintenance of the CSC-like phenotype in tumor cells.

Metabolic features of cancer stem cells: the emerging role of lipid metabolism.

Cancer stem cells (CSCs) are an uncommon subset of tumor cells capable of self-renewal, differentiating, and recreating the parental tumor when transplanted into the murine background. Over the past two decades, efforts toward understanding CSC biology culminated into identifying a set of signaling pathways sustaining "stemness". Nevertheless, while metabolic rewiring is nowadays considered a hallmark of cancer, no consensus has been reached on the metabolic features underlying the plastic nature of CSCs, which are capable of residing in a dormant state, and able to rapidly proliferate when the need to repopulate the tumor mass arises. An emerging concept in the field of CSC metabolism is that these cells are extremely reliant on the activity of enzymes involved in lipid metabolism, such as stearoyl-CoA desaturase 1 (SCD1) and 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR). Indeed, SCD1 and HMG-CoAR have been described as key factors for the correct function of a number of concatenated pathways involved in CSC fate decision, such as Hippo and Wnt. In the present review, we describe metabolic futures of CSCs with a special focus on lipid metabolism, which until now represents an underappreciated force in maintaining CSCs and an attractive therapeutic target.

The effect of targeted therapy on recruited cancer stem cells in a head and neck carcinoma model.

Head and neck cancers (HNC) are known for their repopulation ability driven by cancer stem cells (CSCs). While a small fraction of CSCs proliferates, there are quiescent CSCs that are long-lived and reside outside the cell cycle. Recruitment of quiescent CSCs into the cycle occurs as a response to cell loss and their proliferation may lead to treatment failure. Therefore, CSCs require a more targeted approach to be destroyed. An agent that sensitizes CSC response to treatment is all-trans-retinoic acid (ATRA). The aim of this work is to assess the impact of ATRA combined with radiotherapy on HNC and to analyse the interplay between these agents and cell recruitment. An in silico model is employed to grow a HNC consisting of all cancer cell lineages, with biologically valid kinetic and dynamic parameters. The fate of both cycling and quiescent cancer stem cells is assessed. The Linear Quadratic model is used to simulate radiotherapy, while cellular recruitment and the effects of ATRA on cancer stem cells are modelled based on literature data. A Dose Enhancement Factor (DEF) was determined in order to undertake a quantitative assessment of the effect of ATRA on tumour control. Without recruitment, DEF for the tumour population is 1.06, indicating a slight radiosensitizing effect. Yet, when CSCs are being recruited, the dose enhancement factor is significantly greater (DEF=1.89). Radiation-induced cell arrest and CSC sensitization by ATRA significantly decreases the dose required for CSC eradication in the cycling population. However, the tumour as a whole is not notably affected as the quiescent cells appear to dictate the shape of the survival curve. The model shows that ATRA exhibits a powerful effect on CSCs when combined with radiotherapy. However, the more radioresistant quiescent cell population should not be ignored, as it can be a potential threat to treatment outcome when cells are recruited into the cell cycle.

The role of Notch signaling in gastric carcinoma: molecular pathogenesis and novel therapeutic targets.

Notch signaling, an evolutionarily conserved signaling cascade system, is involved in promoting the progression of different types of cancers. Within the past decades, the Notch signaling pathway has increasingly been shown to have a primary role in deciding the fate of cancer cells and cancer stem cells in the stomach. Most components of Notch signaling are strongly expressed at different levels in gastric carcinoma tissue samples and are associated with a considerable number of clinical parameters. Moreover, crosstalk signaling between the Notch pathway and the Wnt, Ras, and NF-κB pathways promotes the process of gastric carcinogenesis. Consequently, this increases proliferation and prevents apoptosis in gastric cancer cells, and it contributes to the induction of angiogenesis and accelerates the progression of the epithelial-to-mesenchymal transition. Although the Notch signaling pathway presents novel therapeutic targets for cancer therapeutic intervention, there is still a dearth of in-depth understanding of the molecular mechanisms of Notch signaling in gastric carcinoma. In this review, we summarize the landscape of the Notch signaling pathway and recent findings on Notch signaling in gastric cancer. Furthermore, advanced studies and clinical treatments targeting the Notch signaling pathway arediscussed.

Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs

BackgroundEstrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities.MethodsTo better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology.ResultsPhospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate.ConclusionsWe conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

MiR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling

Breast cancer stem cells (bCSCs) have been implicated in tumor progression and therapeutic resistance; however, the molecular mechanisms that define this state are unclear. We have performed two microRNA (miRNA) gain- and loss-of-function screens to identify miRNAs that regulate the choice between bCSC self-renewal and differentiation. We find that micro-RNA (miR)-600 silencing results in bCSC expansion, while its overexpression reduces bCSC self-renewal, leading to decreased invivo tumorigenicity. miR-600 targets stearoyl desaturase 1 (SCD1), an enzyme required to produce active, lipid-modified WNT proteins. In the absence of miR-600, WNT signaling is active and promotes self-renewal, whereas overexpression of miR-600 inhibits the production of active WNT and promotes bCSC differentiation. In a series of 120 breast tumors, we found that a low level of miR-600 is correlated with active WNT signaling and a poor prognosis. These findings highlight a miR-600-centered signaling network that governs bCSC-fate decisions and influences tumor progression.

The Epigenome and Cancer Stem Cell Fate: Connected by a Linker Histone Variant

The molecular features underlying tumor heterogeneity and the role of chromatin components in regulating cell fate within tumors are not well understood. Recently in Science, Torres etal. (2016) showed that the linker histone variant H1.0 functions as a chromatin switch that determines self-renewal versus differentiation decisions in cancer stem cells.

The emerging role of m-TOR up-regulation in brain Astrocytoma.

The present manuscript is an overview of various effects of mTOR up-regulation in astrocytoma with an emphasis on its deleterious effects on the proliferation of Glioblastoma Multiforme. The manuscript reports consistent evidence indicating the occurrence of mTOR up-regulation both in experimental and human astrocytoma. The grading of human astrocytoma is discussed in relationship with mTOR up-regulation. In the second part of the manuscript, the biochemical pathways under the influence of mTOR are translated to cell phenotypes which are generated by mTOR up-regulation and reverted by its inhibition. A special section is dedicated to the prominent role of autophagy in mediating the effects of mTOR in glioblastoma. In detail, autophagy inhibition produced by mTOR up-regulation determines the fate of cancer stem cells. On the other hand, biochemical findings disclose the remarkable effects of autophagy activators as powerful inducers of cell differentiation with a strong prevalence towards neuronal phenotypes. Thus, mTOR modulation acts on the neurobiology of glioblastoma just like it operates in vivo at the level of brain stem cell niches by altering autophagy-dependent cell differentiation. In the light of such a critical role of autophagy we analyzed the ubiquitin proteasome system. The merging between autophagy and proteasome generates a novel organelle, named autophagoproteasome which is strongly induced by mTOR inhibitors in glioblastoma cells. Remarkably, when mTOR is maximally inhibited the proteasome component selectively moves within autophagy vacuoles, thus making the proteasome activity dependent on the entry within autophagy compartment.

MiR-34C Disrupts the Stemness of Purified CD133+ Prostatic Cancer Stem Cells

To find the potential micro-RNA (miRNA) that could determine the fate of prostate cancer stem cells. We compared miRNA expression between our purified CD133+ prostatic cancer stem cells (PCSCs) and CD133- cells. Sphere formation assay and matrigel-based cell invasion assay were applied to determine the stemness of CD133+ PCSCs after our manipulation of miRNA using miRNA mimic or miRNA inhibitor. In this study, we identified that miR-34C was under-expressed in the purified CD133+ PCSCs and enforced introduction of miR-34C attenuated the stemness of CD133+ PCSCs. Clinically, we also observed a negative correlation between miR-34C and CD133. Our data strongly suggest that miR-34C may play essential role in conferring castration resistance by equilibrating PSCS population.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

Imaging Early Fate of Cancer Stem Cells in Mouse Hindlimbs with Sodium Iodide Symporter Gene and I-124 PET.

We investigated the capacity of sodium/iodide symporter (NIS) positron emission tomography (PET) to image and quantitate early engraftment and survival of cancer stem cells (CSCs) in living mice. CT26 colon cancer cells and CSCs were infected with an adenovirus expressing both NIS and enhanced green fluorescent protein (EGFP). Cells were implanted into normal and ischemic hindlimbs of mice, and serial optical and I-124 PET imaging was performed. Extracted tissues underwent I-124 measurements and confocal microscopy. NIS.EGFP gene transfer increased fluorescence and I-124 uptake of CSCs and CT26 cells without adverse effects. I-124 PET clearly visualized implanted tumor cells in vivo, whereas optical imaging was suboptimal. PET revealed 1.95, 2.22, and 1.93-fold greater I-124 uptake by CSC inoculation into ischemic compared to non-ischemic limbs at 2, 15, and 24h, respectively. CT26 cells showed similar but smaller differences. PET findings were confirmed by ex vivo measurements and confocal microscopy. NIS PET can help identify microenvironment conditions that influence early survival of implanted CSCs.

A versatile mathematical work-flow to explore how Cancer Stem Cell fate influences tumor progression.

BackgroundNowadays multidisciplinary approaches combining mathematical models with experimental assays are becoming relevant for the study of biological systems. Indeed, in cancer research multidisciplinary approaches are successfully used to understand the crucial aspects implicated in tumor growth. In particular, the Cancer Stem Cell (CSC) biology represents an area particularly suited to be studied through multidisciplinary approaches, and modeling has significantly contributed to pinpoint the crucial aspects implicated in this theory.More generally, to acquire new insights on a biological system it is necessary to have an accurate description of the phenomenon, such that making accurate predictions on its future behaviors becomes more likely. In this context, the identification of the parameters influencing model dynamics can be advantageous to increase model accuracy and to provide hints in designing wet experiments. Different techniques, ranging from statistical methods to analytical studies, have been developed. Their applications depend on case-specific aspects, such as the availability and quality of experimental data, and the dimension of the parameter space.ResultsThe study of a new model on the CSC-based tumor progression has been the motivation to design a new work-flow that helps to characterize possible system dynamics and to identify those parameters influencing such behaviors. In detail, we extended our recent model on CSC-dynamics creating a new system capable of describing tumor growth during the different stages of cancer progression. Indeed, tumor cells appear to progress through lineage stages like those of normal tissues, being their division auto-regulated by internal feedback mechanisms. These new features have introduced some non-linearities in the model, making it more difficult to be studied by solely analytical techniques. Our new work-flow, based on statistical methods, was used to identify the parameters which influence the tumor growth. The effectiveness of the presented work-flow was firstly verified on two well known models and then applied to investigate our extended CSC model.ConclusionsWe propose a new work-flow to study in a practical and informative way complex systems, allowing an easy identification, interpretation, and visualization of the key model parameters. Our methodology is useful to investigate possible model behaviors and to establish factors driving model dynamics.Analyzing our new CSC model guided by the proposed work-flow, we found that the deregulation of CSC asymmetric proliferation contributes to cancer initiation, in accordance with several experimental evidences. Specifically, model results indicated that the probability of CSC symmetric proliferation is responsible of a switching-like behavior which discriminates between tumorigenesis and unsustainable tumor growth.

Bone microenvironment signals in osteosarcoma development.

The bone is a complex connective tissue composed of many different cell types such as osteoblasts, osteoclasts, chondrocytes, mesenchymal stem/progenitor cells, hematopoietic cells and endothelial cells, among others. The interaction between them is finely balanced through the processes of bone formation and bone remodeling, which regulates the production and biological activity of many soluble factors and extracellular matrix components needed to maintain the bone homeostasis in terms of cell proliferation, differentiation and apoptosis. Osteosarcoma (OS) emerges in this complex environment as a result of poorly defined oncogenic events arising in osteogenic lineage precursors. Increasing evidence supports that similar to normal development, the bone microenvironment (BME) underlies OS initiation and progression. Here, we recapitulate the physiological processes that regulate bone homeostasis and review the current knowledge about how OS cells and BME communicate and interact, describing how these interactions affect OS cell growth, metastasis, cancer stem cell fate and therapy outcome.

SC-31 * RATIONAL DESIGN OF A PI3-KINASE/mTOR INHIBITOR CYTOTOXIC TO BRAIN CANCER STEM CELLS UNDER HYPOXIA

Small molecules that modulate cancer stem cell (CSC) fate and function offer therapeutic potential to improve patient outcome. Malignant gliomas are highly aggressive primary brain tumors with few treatment options and an invariably fatal outcome. High-throughput screening and structure-guided design identified a novel, dual phosphatidylinositol-3-kinase (PI3K)/mammaliam target of rapamycin (mTOR) inhibitor that potently induced apoptosis in glioma cells and glioma patient-derived CSCs. The compound, designated DT61, inhibited PI3K, mTORC1 and mTORC2 kinase activities at nanomolar concentrations in a dose-dependent manner. DT61 dramatically reduced the level of the PI3-K α and β isoforms and led to a reduction in the site-specific phosphorylation and activity of the pro-survival serine-threonine kinase Akt/Protein Kinase B (PKB). DT61 inhibition of PI3K activity prevented the inactivation of glycogen synthase kinase (GSK-3beta) to enhance site-specific phosphorylation and proteasomal degradation of the c-myc oncoprotein that drives gliomagenesis. The apoptotic effect of DT61 on CSCs was synergistically enhanced when combined with radiation treatment, a standard-of-care modality for glioma treatment. Taken together, the results demonstrate that DT61 is a novel, highly effective dual PI3K/mTOR inhibitor that is cytotoxic to brain CSC under normoxia and hypoxia with potential benefit for glioma patients.

SC-35 * BMP SIGNALING REGULATES A QUIESCENT CANCER STEM CELL PHENOTYPE RESPONSIBLE FOR TREATMENT RESISTANCE IN GLIOBLASTOMA

Stem cell niches must be dynamic to enable stem cells to respond promptly and appropriately to physiologic demands. There is growing evidence to suggest that the superfamily of transforming growth factor-βs (TGF-βs) play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems. In glioblastoma, TGF-β has been shown to act as an oncogenic factor through its induction of self-renewal and proliferation in glioma stem cells (GSCs), and inhibition of GSC differentiation. Conversely, treatment of GSCs with BMP4 in vitro was found to direct these cells toward a differentiated, astrocytic phenotype, and abolish their ability to drive tumor growth following xenotransplantation. These studies suggest that TGF-β and BMP signaling direct glioma cells toward an irreversible fate choice, and that maintenance of the cancer stem cell (CSC) phenotype is necessary for tumorigenicity. We postulated that the CSC fate choices in glioblastoma are plastic, and that TGF-β and BMP signaling regulate cell glioma cell dynamics within the tumor microenvironment, as in cellular reprogramming. Further, we postulated that TGF-β and BMP might construe cell identities in glioblastoma that could be relevant to glioblastoma treatment resistance, disease evolution and disease progression. Here, we demonstrate that TGF-β and BMP signaling are active in the glioblastoma microenvironment, and regulate the shuttling of GSCs from an activated to a quiescent phenotype. At the molecular level, this process is mediated, as in EMT/MET, by the ZEB1/miR-200 double-negative feedback loop. Activation of p21 via BMP signaling is one mechanism for resistance to chemotherapy- and radiation-induced cell damage. Quiescent, BMPhi cells then give rise to TGF-βhi GSCs that drive tumour progression. Our findings demonstrate a role for BMP-mediated quiescence in glioblastoma disease resistance and recurrence, and suggest that targeted inhibition of BMP during chemoradiation could favorably alter the natural history of this disease.

Regulated Splicing of the α6 Integrin Cytoplasmic Domain Determines the Fate of Breast Cancer Stem Cells

Although the α6β1 integrin has been implicated in thefunction of breast and other cancer stem cells (CSCs), little is known about its regulation and relationship to mechanisms involved in the genesis of CSCs. We report that a CD44(high)/CD24(low) population, enriched for CSCs, is comprised of distinct epithelial and mesenchymal populations that differ in expression of the two α6 cytoplasmic domain splice variants: α6A and α6B. α6Bβ1 expression defines the mesenchymal population and is necessary for CSC function, a function that cannot be executed by α6Aintegrins. The generation of α6Bβ1 is tightly controlled and occurs as a consequence of an autocrine vascular endothelial growth factor (VEGF) signaling that culminates in the transcriptional repression of a key RNA-splicing factor. These data alter our understanding of how α6β1 contributes tobreast cancer, and they resolve ambiguities regarding the use of total α6 (CD49f) expression as a biomarker for CSCs.