Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Extracellular signal related kinase 3 (ERK3) is one of the atypical mitogen activated protein kinases (MAPK). It is expressed ubiquitously and plays a role in a variety of cellular processes, including cell growth and differentiation. ERK3's role in promoting migration and invasion in various cancers has been well established. ERK3 is upregulated in non-small cell lung cancers (NSCLCs) and has been shown to promote NSCLC tumor growth and progression. However, the regulation of ERK3 in lung cancers remains largely unclear. A recent study indicates that ERK3 phosphorylation at S189, an indicator of ERK3 activity, is upregulated by KRAS in NSCLCs. KRAS is one of the most commonly mutated oncogenes in lung cancers. To study the KRAS dependent regulation of ERK3, knockdown of KRAS was performed and it resulted in a remarkable reduction in ERK3 phosphorylation as well as total ERK3 protein level confirming the regulation of ERK3 by KRAS. Upon knockdown of KRAS a significant reduction of ERK3 mRNA level was observed indicating that KRAS regulates ERK3 at transcriptional level. Further, we found that the regulation of ERK3 by KRAS may be through the transcription factor c-Jun, that is well-known to be activated by KRAS signalling. Our data indicates that c-Jun positively regulates ERK3 transcription in LUAD cell lines. Further, we have found that KRAS upregulates c-Jun activating phosphorylations in LUAD cells, suggesting that KRAS regulates ERK3 through c-Jun. Given the discrepancy regarding the role of ERK3 in NSCLC cell growth reported in previous studies, we have thoroughly investigated the role of ERK3 in cell growth by stable knockdown of ERK3 using shRNA targeting different regions of ERK3 mRNA, as well as by using ERK3 inhibitors in a variety of NSCLS cell lines. While knockdown of ERK3 via targeting the coding region did not affect cell proliferation, targeting the 3'UTR of ERK3 or treatment with ERK3 inhibitors reduced the proliferation of LUAD cells.

Committee: Weiwen Long Ph.D. (Advisor); Kwang-Jin Cho Ph.D. (Committee Member); Michael Craig Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2019, Biomedical Sciences

LKB1 is the fourth-most frequently mutated gene in lung adenocarcinoma, with loss-of-function observed in up to 30% of all cases (Collisson et al. 2014; Kaufman et al. 2014). Our previous work identified a 16-gene signature for LKB1 loss of function through not just mutation, but deletion and epigenetic silencing, which occurs relatively frequently (Lee et al. 2013; Kaufman et al. 2014). We applied this genetic signature to lung adenocarcinoma samples in the Cancer Genome Atlas (TCGA) and discovered a novel association between LKB1 loss and widespread CpG demethylation. LKB1-loss tumors also expressed significantly less DNA methyl transferase (DNMT1) and show depletion of S-Adenosyl-Methionine (SAM-e), which is the primary substrate for DNMT1 activity. Repetitive element transcriptional start sites are demethylated and sensitivity to azacytidine is lower in LKB1 loss. Mechanistically, demethylated CpGs are enriched for FOXA1/2/3 consensus binding sites, and we further identified that FOXA localization and turnover is dependent upon LKB1 and the downstream kinase SIK. Overall, these findings demonstrate that a large number of lung adenocarcinoma patients have a unique epigenetic profile driven by LKB1 loss which could play a role in lung tumorigenicity and resistance to immunotherapy.

Committee: David Carbone (Advisor); Christopher Oakes (Committee Member); Matthew Ringel (Committee Member); Susan Cole (Committee Member); Sameek Roychowdhury (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Genetics; Oncology

Doctor of Philosophy, Case Western Reserve University, 2018, Molecular Medicine

The sustainable activation of the RAS/MAPK and PI3K/AKT signaling pathways in cancer is promoted by a reduction in the activity of the tumor suppressor protein phosphatase 2A (PP2A). Therefore, a novel therapeutic strategy consists of directly activating PP2A, leading to the simultaneous inhibition of these oncogenic pathways. Our lab has successfully developed first-in-class Small Molecule Activators of PP2A (SMAPs), which induce tumor growth inhibition in vivo. Alterations to the putative drug binding site validate PP2A as the direct target of SMAPs. The putative residues of PP2A-Aa that were interacting with SMAPs K194 E197, and L198 were mutated. H358, a KRAS-driven lung adenocarcinoma cell line, was used to create isogenic cell lines stably overexpressing mutated and wild type PP2A-Aa. SMAP response was investigated in vivo using a xenograft model of H358 isogenic cell lines and it was determined that tumors harboring mutant K194R and L198V PP2A-Aa were resistant to SMAPs treatment. Together, our results suggest that residues K194 and L198 are required for drug binding and subsequent target engagement. On another hand, most lung adenocarcinoma (LUAD) patients acquire resistance to tyrosine kinase inhibitors (TKI) via mechanisms enabling the sustained activation of the MAPK and PI3K oncogenic pathways downstream of the tyrosine kinase EGFR. We hypothesize that activation of PP2A simultaneously inhibits the MAPK and AKT pathways and is a promising therapeutic strategy for TKI-resistant LUAD. TKI-resistant LUAD cell lines were treated with SMAPs. RNAseq kinase enrichment analysis followed by principal component analysis indicated that SMAP treatment induces a gene signature similar to a combination of the selective AKT and MEK inhibitors MK2206 and AZD6244, respectively. The therapeutic potential of PP2A activation in vivo was first evaluated in a transgenic mouse model. SMAP- treated mice showed less diffuse lung cancer and a significant decrease in (open full item for complete abstract)

Committee: Goutham Narla (Advisor) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2015, Medicine: Molecular and Developmental Biology

Many cancers are driven by oncogenic K-Ras, yet K-Ras has remained largely undruggable. In this dissertation we explore inhibiting K-Ras signaling by targeting downstream signaling pathways, namely the RhoA and RhoC GTPase pathways. Numerous cellular studies have indicated that RhoA signaling is required for oncogenic Ras-induced transformation. To date very limited data exist to genetically attribute RhoA function to Ras-mediated tumorigenesis in mammalian models. In order to assess whether RhoA is required for K-Ras-induced lung cancer initiation, we utilized the K-RasG12D Lox-Stop-Lox murine lung cancer model in combination with the conditional RhoAflox/flox and RhoC-/- knockout mouse models. We found that deletion of RhoA, RhoC or both did not adversely affect normal lung development. Moreover, we found that deletion of either RhoA or RhoC alone did not suppress K-RasG12D induced lung adenoma initiation. Rather, deletion of RhoA alone increased lung adenoma formation, whereas dual deletion of RhoA and RhoC together significantly reduced K-RasG12D induced adenoma formation. Deletion of RhoA appears to induce a compensatory mechanism that exacerbates adenoma formation. The compensatory mechanism is at least partly mediated by RhoC. These results are in contrast to RhoA knockdown experiments we performed in human lung cancer cell lines, which show dramatic inhibition of malignant phenotypes with RhoA loss. Taken together, this dissertation work suggests that targeting of RhoA alone may allow for compensation and a paradoxical exacerbation of neoplasia, while simultaneous targeting of both RhoA and RhoC is more likely to inhibit oncogenic K-Ras driven lung cancers.

Committee: Yi Zheng Ph.D. (Committee Chair); Vladimir Kalinichenko M.D. Ph.D. (Committee Member); Timothy Lecras Ph.D. (Committee Member); John Morris M.D. (Committee Member); James Mulloy Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. Ph.D. (Committee Member) Subjects: Surgery

PhD, University of Cincinnati, 2019, Medicine: Systems Biology and Physiology

Disruption of circadian rhythm can lead to serious sleeping disorders and predispose to a number of life-threatening diseases, including cancer. Circadian splicing adds an additional regulatory layer to the time keeping mechanism in plants, flies and mammals. The circadian regulation of alternative splicing, including that of core clock genes themselves, are speculated to be a central mediator of clock function, however, no comprehensive analyses of this mechanism exist to date. To develop an improved understanding of circadian splicing in mammals I describe a series of comprehensive analyses of circadian splicing within and across diverse healthy mouse, baboon and human tissues, spanning thousands of samples. These analyses confirm that conserved tissue-specific and tissue-shared circadian splicing events (CSEs) are frequent and can be identified from multiple study designs and recommend workflows for accurate circadian splicing analysis. Our analysis demonstrates a higher number of tissue-specific CSEs compared to circadian gene expression. Transcriptionally, temperature sensitive and other circadian splicing factors (SFs) are also found rhythmic in majority of the tissues. Cross-tissue CSEs frequently contain binding sites for these circadian SFs likely targeting specific CSEs and regulating splicing at the peripheral tissue level. Notably, these evolutionarily conserved CSEs and pan-tissue CSEs frequently impact prior defined cancer regulators, RNA binding proteins previously implicated in thermoregulation and splicing auto-regulation. I demonstrate the importance of these circadian findings specific in Lung cancer, which suggest the existence of novel putative chronotherapeutic targets. To enable the broad research community, we have developed an easy-to-use online web-portal to explore and compare these results across species. As such, these data have the potential to highlight intriguing new roles for splicing regulation in normal circadian biology.

Committee: Nathan Salomonis M.D. (Committee Chair); Christian Hong Ph.D. (Committee Member); Jaroslaw Meller Ph.D. (Committee Member); Yana Zavros Ph.D. (Committee Member); Tongli Zhang Ph.D. (Committee Member) Subjects: Bioinformatics