Browsing by Author "Yao, TingTing, committee member"
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Item Open Access Autophagy modulation: role in anti-cancer therapy(Colorado State University. Libraries, 2015) Barnard, Rebecca A., author; Gustafson, Daniel L., advisor; Thamm, Douglas H., committee member; Thorburn, Andrew, committee member; Yao, TingTing, committee memberAutophagy is a conserved lysosomal degradation process characterized by cellular self-digestion. Autophagy results in turnover of the cytoplasm allowing for metabolic maintenance and organelle quality control, particularly during cell stress. These aspects of autophagy can facilitate tumor cell survival and resistance. As such, autophagy inhibition is being explored in clinical trials as a novel approach to chemosensitization. However, there are still a number of unresolved concerns in regards to the use of autophagy inhibition as a therapy. It is still unclear how autophagy functions in metastasis development. Therefore, we investigated the role of autophagy in metastasis by modulating autophagy in different mouse models and cell based assays that reflect the steps of metastatic development. We found that autophagy was not required for tumor cell colonization within the site of metastasis nor did autophagy alter the metastatic capabilities of the cells. Rather, autophagy appeared to impact the pre-metastatic environment through effects on bone marrow derived cell number which mediate the establishment of the metastatic niche. Stimulating autophagy, before tumor cells disseminated, could speed metastatic development and increase the number of these cells within circulation and eventual sites of metastasis. Correspondingly, inhibiting autophagy could delay metastasis and reduce circulating bone marrow derived cells. These studies suggest that autophagy is most critical in the stages prior to tumor cell arrival at the site of metastasis, by influencing the metastatic microenvironment. While increased autophagy is often considered to be a common tumor adaptation, it is now apparent that some tumor types are more dependent on autophagy than others. However, it is not well understood which tumors these are. Triple negative, Stat3 activated breast cancers were identified as autophagy dependent by collaborator Dr. Paola Maycotte. We tested the efficacy of autophagy inhibitor chloroquine (CQ) in xenograft models of triple negative and estrogen receptor positive breast cancer. CQ was only efficacious in the triple negative tumors. As some canine osteosarcomas also have constitutive Stat3 activity, we assessed the relationship of Stat3 activity and CQ sensitivity. Unlike in breast cancer, Stat3 phosphorylation did not indicate increased sensitivity to CQ in canine osteosarcoma. However, all the osteosarcoma cell lines responded to treatment. Using microarray analysis we identified potential compensatory pathways that have been previously reported to work in concert with autophagy in other cell types and may serve as useful combinational therapies. Currently, the only autophagy inhibitor available clinically is CQ or derivative hydroxycholorquine (HCQ). It is still uncertain whether these drugs can actually achieve autophagy inhibition in patients. Dogs serve as a good model for human cancer and there is an unmet need for novel therapies in the treatment of canine lymphoma. Thus we conducted a phase I clinical trial in canine lymphoma patients with the goals of finding a maximum tolerated dose in combination with doxorubicin (DOX) and the relationship of HCQ concentration and autophagy inhibition. We found that this combination can be well tolerated with a 20% reduction in DOX. HCQ can achieve autophagy inhibition in patients, but not consistently. There appears to be a threshold requirement of HCQ needed in order to effectively inhibit autophagy. There was a suggestion of efficacy as response rate was superior to historical data employing DOX alone. Therefore autophagy inhibition warrants further clinical study as an anti-cancer therapy.Item Open Access Clinical importance of autophagy dependency and inhibition in cancer treatment(Colorado State University. Libraries, 2021) Van Eaton, Kristen M., author; Gustafson, Daniel L., advisor; Munsky, Brian, committee member; Thamm, Douglas, committee member; Thorburn, Andrew, committee member; Yao, TingTing, committee memberAutophagy, a lysosomal degradation recycling process, has a complex and context-dependent role in cancer. Certain cancers have been found to be inherently dependent on autophagy for survival regardless of the environment. Autophagy is also implicated as a mechanism of resistance to many chemotherapies. More autophagy dependent tumors are generally more sensitive to autophagy inhibition genetically and pharmacologically. Therefore, determining what tumors are autophagy dependent is important for selecting patients that are viable candidates for autophagy inhibition. Currently, autophagy inhibition is being tested in over 90 clinical trials using FDA- approved hydroxychloroquine (HCQ) alone or in combination with other therapies. However, responses have been variable, especially in trials where HCQ is used as a monotherapy. Further, the relationship between HCQ pharmacokinetics and pharmacodynamics is not well understood in patients. Pharmacokinetics of HCQ and one of its active metabolites DHCQ was assessed in non-tumor bearing mice. Both parent and metabolite were observed at clinically relevant concentrations after 72 hr and this corresponded with evident autophagy inhibition in various tissues, although autophagy inhibition was inconsistent across the mice. The pharmacokinetic data established 60 mg/kg as the human equivalent dose observed in patients based on HCQ exposure. Cellular responses to HCQ were assessed in 2D cell culture, 3D tumor organoids, and in vivo tumor xenografts using autophagy dependent and autophagy independent tumors. Overall, cellular responses were similar across the in vitro and in vivo methods. Autophagy was inhibited regardless of autophagy status, but autophagy dependent tumors had increased cell death and decreased cell proliferation at earlier time points and lower doses of HCQ, suggesting autophagy dependency matters for optimal results. Since autophagy inhibition was inconsistent in vivo, it is still important to determine better biomarkers and possibly consider using more potent autophagy inhibitors in the clinic. Since there have not been any major advancements in osteosarcoma survival over the past four decades, autophagy dependency was assessed in osteosarcoma. Osteosarcoma was found to be intermediately to very dependent on autophagy following a genetic screen. Further, initially autophagy dependent tumor cells were able to survive and adapt to autophagy loss. Not all tumor cells adapted in the same way nor were these autophagy deficient tumor cells more sensitive to standard osteosarcoma chemotherapy, highlighting the difficulty of determining what context autophagy inhibition should be used in the clinic. Since some autophagy inhibitors like HCQ are lysosomal inhibitors and do not specifically target autophagy alone, the results of these studies also emphasized the importance of understanding whether autophagy inhibition via lysosomal degradation or autophagy inhibition of the autophagic pathway itself is superior. Overall, these results indicate targeting autophagy in osteosarcoma is a promising therapy.Item Open Access Parsing PARP: the enzymatic and biophysical characterization of poly (ADP-Ribose) polymerases I and II(Colorado State University. Libraries, 2015) Hepler, Maggie R. D., author; Luger, Karolin, advisor; Bailey, Susan, committee member; Yao, TingTing, committee memberThe ADP-ribosyl transferase (ART) family is a prominent group of at least seventeen enzymes comprised of mono (ADP-ribose) transferases (MARTs) and poly (ADP-ribose) polymerases (PARPs). Each family member contains a conserved PARP signature motif in the catalytic domain. Enzymatically active proteins, in the presence of co-factor NAD+, catalyze individual or multiple ADP-ribose groups onto themselves or other proteins in automodification and heteromodification, respectively. The act of ADP-ribosylation implicates the ART family in a multitude of cellular processes including, but not limited to, transcription, apoptosis, DNA damage, metabolism, and inflammation. The founding member of the ART family is PARP-1, a first responder to DNA damage and regulator of active gene expression. In its inactive state and as a chromatin architectural protein, PARP-1 tightly binds chromatin, thereby regulating cellular activities, signifying the importance of PARP-1 and chromatin interaction. Importantly, PARP-1 must be activated and automodified in order to bind histones and gain nucleosome assembly function. Structurally similar and in many ways thought to be functionally redundant, PARP-2 is also thought to primarily function in the DNA damage response. PARP-2 has a non-canonical DNA binding domain, and therefore it is able to recognize different types of DNA structures in comparison to PARP-1, which could suggest a unique role for PARP-2 in repair. PARP-2 has not been extensively studied in a chromatin or gene regulation context due to this assumed redundancy. Given the pronounced functional changes in PARP-1 upon automodification, it is important to better understand what exactly triggers its enzymatic activity. Similarly, due to the functional redundancy of PARP-2, insight into activators of its enzymatic activity could indicate specificity and selectivity for the protein. However, determining the details of nuclear components that activate PARP-1 and PARP-2 are limited by the availability of a reliable quantitative and kinetic assay, as well as by the availability of defined substrates. These limitations hinder the separation of potent, and thus biologically relevant, activators from weak or non-specific activators. Utilizing a fluorescence based enzyme assay adapted for this system, kinetic parameters of PARP-1 and PARP-2 allosteric activators are reported here. As proof of principle and to test the reliability of the enzymatic assay, PARP-1 and PARP-2 activity was first tested with nucleic acids and other previously reported activators, such as nucleosomes and histones. Next, potentially novel activators were tested. Notably, PARP-1 is activated in the presence of its enzymatic product, PAR, indicating a mechanism by which PARP-1 could spread at sites of DNA damage and active gene expression. PARP-2 exhibits unique activation and specificity different from that of PARP-1 through its enzymatic preference for RNA. Further, PARP-1 remains the prominent chromatin related PARP due to the weak interaction, both activity and affinity, of chromatin with PARP-2. However, while PARP-1 and PARP-2 can act individually, affinity and activity studies demonstrate a PARP-1 and PARP-2 complex suggesting that these proteins can act sequentially and simultaneously with one another during a PAR-mediated recruitment and signaling cascade. Overall, these data indicate novel functions and mechanisms for PARP-1 and PARP-2 within the nucleus as critical responders to DNA damage and gene regulation.