Browsing by Subject "Neoplastic Stem Cells"
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Item Cellular Mechanisms of Cancer Stem Cells(2008-05-12) Bhagwandin, Vikash Jaganath; Shay, Jerry W.The concept of cancer stem cells is based on the presence of adult stem cells within tissues that can transform and give rise to tumors that also retain stem-like properties. Although advanced solid human tumors are treated with chemotherapy and/or radiotherapy, in most cases there are only partial responses and tumors generally recur. These observations suggest that here is a resilient 'rare' cancer cell type that survives therapy, which gives precedence to the following cancer stem cell hypothesis. Hypothesis: Cancer cells retain stem-like characteristics that may reflect their degree of malignancy and resistance to conventional therapy. The experiments presented in this thesis report focus on identification of cancer stem cells by exploiting conserved cellular mechanisms of normal stem cells such as; label retention (LRC) and transit amplification (TAC). Panc-1 human pancreatic cancer cells pulsed with BrdU gave rise to tumors that contain a 'rare' quiescent population of cells retaining their BrdU label up to one month in vivo. Transit amplification of these cells was explored in vitro by using a stem cell marker, the 'side population' SP), that gave rise to two distinct cell populations after one week in culture. Isolated Panc-1 clones with varying degrees of the SP phenotype and metastatic potential demonstrated that loss of SP coincides with increased metastasis. Treatment of these clones with the chemotherapeutic drug gemcitabine showed that the presence of SP and decreased metastatic potential conferred resistance. Combined treatment of a telomerase inhibitor (GRN163L) and gemcitabine sensitized cancer stem cells in vitro. These findings suggest that cancer stem cells may retain stem-like characteristics such as label retention, symmetric and asymmetric divisions, and drug sensitivity to govern tumor progression and resistance. These finding may in the future allow the dissection of underlying molecular mechanisms regulating cancer stem cells and provide opportunities to discover therapies that specifically target these cancer stem cells.Item Examining the Regulation and Function of Cancer Testis Antigens(2015-12-01) Pavlovsky, Ashly Ann; Brekken, Rolf A.; Whitehurst, Angelique Wright; Johnson, Jane E.; Cobb, Melanie H.Cancer Testis Antigens (CTAs) are a class of genes whose expression is generally restricted to the testis, but are reactivated in cancer cells. The function and regulation of many CTAs are unknown, however several CTAs have been shown to impact tumor cell fitness and correlate with poor prognosis. Our lab became interested in CTAs after a pan-genomic-loss-of-function RNAi screen identified several CTAs as chemo-sensitizers. Acrosin Binding Protein (ACRBP) was identified in this screen and further analysis confirmed ACRBP's function as a microtubule stabilizer that protects cancer cells from the mitotic defects attributed to paclitaxel treatment. Because ACRBP is not expressed in normal somatic tissue, we are interested in how ACRBP is reactivated in cancer cells. In mouse spermatogonium, the ACRBP promoter is bound by Cyclic-amp Response Element Modulator (CREM) at a conserved Cyclic-amp response element (CRE). Depletion of its close family member CREB results in a loss of ACRBP expression in cancer cells, while overexpression of CREB induces ACRBP expression. Interestingly, this regulation appears to be phospho-independent and seems to apply to other CTAs. Another CTA, Chondrosarcoma Associated Genes 1, was identified by a loss-of-function RNAi proliferation screen as a supporter of melanoma cell proliferation. CSAG1 is highly expressed in Melanoma lines with little to no expression in normal tissues. Additional CSAG1 siRNA experiments have validated the screen data confirming CSAG1's involvement in melanoma cell proliferation. Additionally, CSAG1 loss-of-function reduces long-term melanoma cell viability and induces senescence in cancer cells. Consistent with this data, overexpression of CSAG1 enhances colony-forming ability in cancer cells. These data suggest that CSAG1 supports cancer cell viability. Further studies will help elucidate how CSAG1 supports tumor cell fitness. Understanding the regulation and function of CTAs may provide new insight into novel cancer therapeutics.Item Histone Demethylase LSD1 Restricts the Size of the Germline Stem Cell Niche in Drosophila Ovaries(2013-01-16) Eliazer, Susan; Buszczak, MichaelSpecialized microenvironments called niches keep stem cells in an undifferentiated and self-renewing state by producing a variety of factors. The size and signaling output of niches must be finely tuned to ensure proper tissue homeostasis. I use the Drosophila female germline as an excellent model system to study niche development and function. Five to seven somatic cap cells form the ovarian stem cell niche and produce dpp, a BMP homolog necessary for the maintenance of germline stem cells (GSCs). Mutations in Lsd1, a histone demethylase exhibit GSC-like tumor formation. Clonal analysis, cell-type specific knock down and rescue experiments demonstrate that Lsd1 functions within the escort cells that reside immediately adjacent to cap cells (niche). Loss of Lsd1 causes the escort cells to adopt an intermediate fate expressing both escort cell and cap cell markers and enables them to function as ectopic niches for the expanded stem cell population. Temporally restricted gene knock-down experiments suggest that Lsd1 functions both during development, to specify EC fate, and in adulthood, to prevent ECs from forming ectopic niches independent of changes in cell fate. Lsd1 specifically functions to repress dpp, the niche signal in the adult germaria. I have identified engrailed as a direct target of Lsd1 by performing Chromatin Immunoprecipitation (ChIP-seq) analysis in the escort cells of the Drosophila ovary. Engrailed is expressed in the cap cells of wild type germaria and in Lsd1 mutants engrailed transcripts are misexpressed in the escort cells. Knocking down engrailed expression in the escort cells suppresses the Lsd1 mutant phenotype. Moreover, ectopic expression of engrailed in the escort cells displays a GSC-tumor phenotype. Furthermore, I have shown that Engrailed functions upstream of dpp, and activates its expression in the cap cell niche.Item Telomere Length Studies in Human Cancer Cells(2011-08-10) Buseman, Christen Marie; Shay, Jerry W.Telomeres consist of repetitive DNA sequences and their associated binding proteins, and serve to protect linear chromosome ends from being recognized as double stranded breaks in need of repair. The telomeres of most normal diploid cells shorten with every cell division until they reach a critically short length, at which time the cells undergo senescence or apoptosis. Cancer cells which have the ability to divide indefinitely must prevent their telomeres from becoming critically short, and the majority of cancer cells achieve this by upregulating telomerase. Maintaining telomere length involves regulating the dynamic between telomere shortening and telomere elongation. However, there are still many aspects of this dynamic regulatory process that are unknown. Many methods of telomere length assessment have been developed that utilize a variety of molecular techniques, but a major shortcoming of these methods is that they lack the ability to detect single short telomeres that are thought to trigger replicative senescence. Thus, the objective of this work was to develop an assay, named Universal STELA, which can generate an accurate distribution of telomere lengths on all chromosomes and allow for the study of the shortest telomeres in experimental settings. Universal STELA was first used to determine if cancer stem cells are susceptible to telomerase inhibition therapy because they have a larger fraction of shorter telomeres than non cancer stem cells. Cancer stem cells are thought to contribute to cancer metastasis and recurrence, and therapies like telomerase inhibition that target cancer stem cells may lead to more durable treatment outcomes. Universal STELA was next used to investigate regulation of telomerase action. C- and G-STELA were used to determine that telomerase activity is coupled to telomere replication, while C-strand fill-in is delayed until S/G2. Universal STELA was used to compare the rate of elongation of short, average and long telomeres when telomeres are shorter than their maintenance length.