Browsing by Subject "Cancer stem cell"

Human cancer is a leading cause of morbidity and mortality in the developed world. Contrary to the classical model in which tumors are homogeneously composed of malignant cells, accumulating evidence suggests that subpopulations of highly malignant cells play a dominant role in tumor initiation and growth. These cells have the capacity for prolonged self-renewal and they efficiently generate tumors that phenotypically resemble the parental tumor in transplantation assays. Such characteristics are reminiscent of normal stem cells, and these potently tumorigenic cells have therefore been called cancer stem cells (CSCs). Importantly, studies have shown that CSCs are central mediators of therapeutic resistance, tumor angiogenesis, and metastatic or invasive potential. In the case of malignant glioma, poor patient survival and the paucity of effective therapeutic advances have been attributed to inherent CSC growth potential and treatment resistance, respectively. For this reason, there is great interest in elucidating the molecular features of CSCs, with the ultimate hope of developing CSC-directed therapies.

Given the overlap between the highly malignant characteristics exhibited by CSCs and those promoted by the PI3K/AKT pathway, we hypothesized that AKT activity within CSCs could represent a reasonable therapeutic target for CSC-directed therapies. Indeed, a pharmacological inhibitor of AKT preferentially targeted glioma CSCs versus non-CSCs and was associated with increased apoptosis and impaired tumorigenesis. These data suggest that interventions targeting AKT could effectively target glioma CSCs.

Quite distinct from the PI3K/AKT pathway, we hypothesized that the pro-survival and pro-growth features of nitric oxide (NO) might also operate in glioma CSCs. Our experiments found that glioma CSCs produced more NO than non-CSCs, which is attributed to inducible nitric oxide synthase (iNOS) expression and activity within the CSCs. Interference with iNOS activity or expression, as well as selective NO consumption, attenuated CSC growth and tumorigenicity. The mechanism behind iNOS-mediated survival appears to involve, at least in part, suppression of the cell cycle inhibitor CDA1. iNOS inhibition decreased glioma growth in murine xenografts and human expression studies demonstrate an inverse correlation between iNOS expression and patient survival.

To more fully evaluate the biological effects of NO in CSCs, we designed a novel strategy to consume NO within mammalian cells through heterologous expression of E. coli flavohemoglobin (FlavoHb). This enzyme is a highly specific NO dioxygenase which converts NO to inert nitrate several orders of magnitude faster than iNOS synthesizes NO. Expression of FlavoHb in mammalian cells is therefore a novel and functional tool to interrogate the role of NO in cellular stress and signaling.

In summary, this doctoral thesis focuses on several molecular characteristics that define malignant CSCs and describes a novel strategy for studying NO, which is one of the CSC-specific molecular effectors.

Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.

Ovarian cancer is the fifth leading cause of cancer-related death in the United States and the most lethal gynecologic malignancy. Patients with ovarian cancer are generally diagnosed at stage III or IV, when ascites fluid becomes a common symptom. The volume of ascites positively correlates with the extent of ovarian cancer metastasis and negatively with prognosis; however, the mechanisms explaining their effect are unknown.

We hypothesize that ascites enriches for cancer stem-like cells. Our present study demonstrates that mice injected with ID8 cells, a murine epithelial ovarian cancer line, have remarkably shortened survival, when injected together with ascites supernatant derived from tumor-bearing mice. Moreover, compared to their counterparts cultured in regular medium, ID8 cells cultured in ascites fluid, or isolated directly from ascites, show an increased expression of stem cell markers Oct4 and CD133. These cells also exhibit enhanced self-renewing ability in sphere assay, suggesting that ascites enriches for stem-like cells.

Furthermore, we demonstrate that ascites enriches for cells expressing cell surface GRP78, a stress-inducible endoplasmic reticulum chaperone which also appears on the plasma membrane (memGRP78) of aggressive cancers. MemGRP78 + cells correlate with stem cell properties of self-renewal and tumor initiation, suggesting GRP78 is a novel stem cell marker. Importantly, antibodies against the COOH terminal domain of GRP78 significantly reduce the self-renewing ability of murine and human ovarian cancer cells pre-incubated with ascites.

In conclusion, our study demonstrates that ascites enriches for stem-like cells in ovarian cancer cell lines. Furthermore, the inhibitory effect of antibodies against the COOH terminal domain of GRP78 suggests that memGRP78 is a logical therapeutic target for ovarian cancer.

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1) is an ATP-dependent DNA-annealing helicase that reverses stalled replication forks. Its loss of function genetic alterations occurs in a subset of glioblastomas (GBMs) and has been found to be associated with alterative lengthening of telomeres (ALT+) in tumor cells. ALT tumors exploit homologous recombination to maintain telomere length and share common characteristics, including compromised telomere shelterin protein and increased replication stress in the telomere region.We established a SMARCAL1-null, ALT+ primary GBM culture. We show that the primary GBM culture displays stable ALT features and maintains mostly consistent karyotypes after their growth in mice. Transcriptomic profiling of the ALT+ primary GBM cells that have been propagated in vitro and those that have undergone propagation in vivo revealed the effects of microenvironments on the gene expression of these tumor cells. By using a doxycycline-inducible expression system, we show that the ALT+ features in the GBM primary culture can be turned off and on by the restoration or withdraw of exogenous wild-type SMARCAL1, but not its enzymatic dead mutant counterpart. Telomere pull down assays demonstrated the expression of SMARCAL1 effectively attenuates the process of ALT in tumor cells. In supporting the critical roles of ALT for tumor progression, induced restoration of wild-type SMARCAL1, but not its enzymatic dead mutant, effectively suppresses tumor progression in vivo. By taking advantage of our well characterized ALT model system, we are investigating the role of intrinsic DNA damage in tumorigenesis. Intrinsic DNA replicative stress occurs constantly in tumor cells. However, the pathogenic ramifications of replicative stress and the strategies cancer cells undertake to adapt remain to be fully defined. Here, we attempt to address these questions, using isogenic sarcoma and glioma cell line models differing in their intrinsic telomeric replicative stress levels, we show that intrinsic replicative stress promotes cancer stemness in human sarcoma and glioma cells. Further, molecular profiling analysis of human gliomas supports that human gliomas with higher levels of intrinsic replicative stress levels have increased stemness. We show this intrinsic replicative stress-stimulated stemness is accompanied by nonrandom segregation of chromosomes in mitotic cells. More notably, this nonrandom chromosome segregation is associated with asymmetric partition of CD133, a canonical marker for cancer cell stemness, in that the newly synthesized set of chromosomes are placed in one progeny cell while the set serving as templates for DNA replication turns to co-segregate with CD133 in another. We further reveal that this asymmetric co-segregation of chromosomes and CD133 depends on the Wnt/β-catenin signaling pathway. Collectively, these findings identify intrinsic DNA replicative stress as a driver of cancer cell stemness, and suggest a coordinated, Wnt/β-catenin signal-driven process of asymmetrically partitioning DNA and proteins in these cells, potentially as a way of maintaining cellular heterogeneity and population fitness in response to DNA damage. They also highlight and provide new insights into the roles of the Wnt/β-catenin pathway in maintaining tumor cells stemness, and suggest strategies for therapeutically targeting DNA damage-driven stemness in gliomas.