AUTOPHAGY AND CANCER: THE ROLE OF AUTOPHAGY IN THE HYPOXIC TUMOR MICROENVIRONMENT AND TRANSLATIONAL IMPLICATIONS OF TARGETING ULK1 IN NEUROBLASTOMA
Open Access
- Author:
- Dower, Christopher Micheal
- Graduate Program:
- Molecular Medicine
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 23, 2018
- Committee Members:
- Hong-Gang Wang, Dissertation Advisor/Co-Advisor
Hong-Gang Wang, Committee Chair/Co-Chair
Rosalyn Bryson Irby, Committee Member
Scot R Kimball, Committee Member
Charles H Lang, Outside Member - Keywords:
- Autophagy
Cancer Metastasis
Hypoxia
Tumor Microenviroment
Neuroblastoma
ULK1 - Abstract:
- A solid cancerous tumor is a complex mixture of many different cell types, varying compositions of extracellular matrix, and fluctuating degrees of oxygen and nutrient availability. This heterogeneity generates distinct stress-inducing tumor microenvironments (TME) that add selective pressure on cancer cells, selecting aggressive cancer cells that have an advantage of survival or metastatic potential. To survive stressful TMEs, cancer cells utilize an evolutionary conserved intracellular degradation and recycling mechanism, called autophagy (from the Greek for “self-eating”). Autophagy uses double membraned vesicles, called autophagosomes, to engulf cytoplasmic cargo for delivery to the lysosome for degradation. In general, autophagy is thought to promote cancer cell survival by facilitating the degradation of various cytoplasmic components and recycling of essential nutrients in starvation conditions, making it an attractive therapeutic strategy for treating cancers. However, the precise role of autophagy within the TME is controversial, as it exhibits both tumor-promoting and tumor-suppressing phenotypes, making it difficult to conclude whether autophagy is a viable target for cancer therapy. In particular, this paradox is most evident in regards to the role of autophagy in regulating cancer metastasis, as reports conflict as to whether autophagy is truly a metastasis-suppressing or -promoting pathway. Emerging research indicates that the influence of autophagy on cancer progression is dependent on several factors, including cancer cell type and the TME. Thus, more physiologically relevant assessments which incorporate TME-associated stress and cell-to-cell interactions are needed to better understand the role of autophagy in tumor progression. Accordingly, the primary focus of this doctoral dissertation involves elucidating the role of autophagy in tumor progression in the context of hypoxia (i.e. low oxygen conditions), a hallmark of the TME. In chapter 2, we explored the impact of autophagy on the pathophysiology of breast cancer cells, using a novel hypoxia-dependent, reversible dominant negative strategy to regulate autophagy at the cellular level within the TME. Suppression of autophagy via hypoxia-induced expression of the kinase-dead dominant-negative mutant of ULK1 (dnULK1K49R) increased lung metastases in MDA-MB-231 xenograft mouse models. Consistent with this effect, expressing a dominant-negative mutant of ULK1 or ATG4b or a ULK1-targeting shRNA facilitated cell migration in vitro. Functional proteomic and transcriptome analysis revealed that loss of hypoxia-regulated autophagy promotes metastasis via induction of the fibronectin integrin signaling axis. In agreement, loss of ULK1 function increased fibronectin deposition in the hypoxic TME. Additionally, we report that low expression of autophagy genes predicts a worse prognosis in human breast cancer. Together, our results indicated that hypoxia-regulated autophagy suppresses metastasis in breast cancer by preventing tumor fibrosis. These results also suggest caution in the development of autophagy-based strategies for cancer treatment. In addition to this, a secondary focus of this dissertation involved examining the translational implications of targeting autophagy for therapeutic benefit in neuroblastoma, a cancer of immature nerve cells that predominantly effects children under five years old. In chapter 3, we demonstrate that targeted inhibition of an essential autophagy kinase, ULK1, with a recently developed small molecular inhibitor of ULK1, SBI-0206965, significantly reduces cell growth and promotes apoptosis in SK-N-AS, SH-SY5Y, and SK-N-DZ neuroblastoma cell lines. Furthermore, inhibition of ULK1 by a dominant-negative mutant of ULK1 (dnULK1K49R) significantly reduced growth and metastatic disease and prolonged survival of mice bearing SK-N-AS xenograft tumors. We also show that SBI-0206965 sensitized SK-N-AS cells to TRAIL treatment, but not mTOR inhibitors (INK128, Torin1) or topoisomerase inhibitors (doxorubicin, topotecan). Collectively, these findings demonstrate that ULK1 is a viable drug target and inhibitors of ULK1 may provide a novel therapeutic option for the treatment of neuroblastoma. Furthermore, this work demonstrates the antitumor effects of targeting an essential autophagy gene in neuroblastoma mouse models. In summation, this dissertation has elucidated that hypoxia-regulated autophagy acts to suppress metastasis in breast cancer, and demonstrated that ULK1 kinase is a viable drug target for the treatment of neuroblastoma. Collectively, this work has further defined the complex role of autophagy in tumor biology, as well as provided pre-clinical data that may aid in the development of novel treatment options for cancer patients.