Date of Award

5-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmaceutics

Research Advisor

Meena Jaggi, Ph.D.

Committee

Santosh Kumar, Yi Lu, Murali M Yallapu, Nadeem Zafar

Abstract

Pancreatic cancer ranks as third deadliest cancer worldwide in both men and women. The lack of diagnostic methods, chemo-resistance and invasiveness of the disease diminishes the overall survival rate in patients. Dysregulated glucose metabolism and several molecular determinants including oncogenic signaling pathways play pivotal roles in the development, progression and metastasis of pancreatic cancer. The aberrant glucose metabolism is the most prevalent key factor that influences pancreatic tumorigenesis. The cancer cells of pancreas utilize oxygen for the breakdown of glucose to lactate, which is known as Warburg effect. This phenotype of pancreatic cancer encourages proliferation, growth, migration and invasive phenotypes of cancer cells that worsens the disease. The upstream regulatory molecules responsible for deviant activity of glucose transporters during aberrant glucose metabolism was not very well defined. Thus, identification of new molecular determinants that regulate glucose metabolic pathway would provide promising therapeutic outcomes. Our lab has established the role of MUC13 protein in pancreatic cancer development. Another interesting molecule, Protein Kinase D1 has been reported to play essential role in hypoxic metabolism in squamous cell carcinoma, and glucose uptake in cardiac hypertrophy. Also, it has been extensively studied in our research group, and has been suggested by some other groups to have an essential role in pancreatic cancer. However, its role in regulating glucose metabolism in pancreatic cancer remains elusive. In this dissertation, we investigated that MUC13 and PKD1 proteins are involved in abrupt glucose metabolism in pancreatic cancer cells. We have demonstrated that targeted overexpression of these oncogenes upregulates key oncogenic signaling components involved in aberrant glucose metabolism in pancreatic cancer cells. We also investigated a natural agent Steviol that restored glucose metabolism in pancreatic cancer cells via repression of MUC13 and PKD1 expression. MUC13 is a transmembrane glycoprotein which has an elevated expression in pancreatic cancer. Due to the presence of a cytoplasmic domain, it confers kinase activity and regulates many signaling processes. Therefore, we investigated the effect of MUC13 in metabolic reconditioning of pancreatic cancer. As a result, it was observed that presence of MUC13 in pancreatic cancer upregulated glucose uptake and lactate secretion. This in turn led to more aggressive behavior of the tumor, as evidenced by enhanced proliferation, migration and invasive characteristics of the cells as compared to MUC13 null cells. Interestingly, we observed the interaction of MUC13 with Glut-1 protein, which influences glucose uptake in cancer cells. This was confirmed through various assays such as: immunoprecipitation, immunofluorescence, co-capping and proximity ligation assay. The interpretation of inhibitor results envisaged the role of NFҡB pathway during the molecular interactions of MUC13 and Glut-1. To understand the clinical importance of this mechanism, we evaluated this interaction in tumor samples from the patients with pancreatic disease in advanced stages. Our findings fortified the mechanistic role of MUC13 in rewiring of aberrant glucose metabolism. Altogether, we believed that MUC13 has clinical implication as a key molecule that might be responsible for dysregulated glucose metabolism. As discussed earlier about the paucity of associated proteins related to dysregulated glucose metabolism, we further investigated the possible key players with kinase domain for association and activation with other signaling molecules.
Protein Kinase D1 (PKD1), a Ser/Thr kinase has been shown to be involved in progression of pancreatic cancer. In this study, we established PKD1 as a novel molecular target and its involvement in regulating aberrant glucose metabolism in pancreatic cancer. We observed that PKD1 was overexpressed in pancreatic cancer tissues compared to normal human tissues. We also found constitutive expression of PKD1 in various pancreatic cancer cells as compared to normal pancreatic ductal epithelial cells. The overexpression of PKD1 in low PKD1 expressing pancreatic cancer cells enhanced tumorigenic characteristics. We observed that specific knockdown of PKD1 inhibited key oncogenic signaling components in pancreatic cancer cells suggesting its oncogenic role in pancreatic cancer. Additionally, during the PKD1 overexpression, cells displayed increased glucose consumption and lactate production. This suggested role of PKD1 in metabolic reprogramming in pancreatic cancer. Also, we observed that PKD1 stimulates the glucose uptake through mTORC1, a component of mTOR signaling pathway. Additionally, the knockdown of PKD1 promoted the chemosensitivity of cells towards gemcitabine and 2DG (2-deoxyglucose). Overall, these results indicated a significant function of PKD1 in rewiring of glucose metabolism in pancreatic cancer. It was mandating to target and counteract the aberrant glucose metabolism along with the proteins identified in our previous results. In this perspective herein, we proposed steviol, a natural sweetener from plant origin to assess its inhibitory action towards dysregulated glucose metabolism and its associated proteins (MUC13 and PKD1). The reason behind steviol selection for this approach includes that it mimics the glucose molecule that facilitates enhanced uptake of drug (steviol) within the cells and on the other side, it provides glucose homeostasis to adjacent normal cells. These features made steviol as dual functional drug by overtaking other natural drugs for promising results. The exposure of steviol inhibited the proliferative (IC50: 10mM) and clonogenic abilities of pancreatic cancer cells. It also suppressed the cell migration and cell invasion capabilities of pancreatic cancer cells. Further, steviol caused the selective inhibition of intracellular glucose intake and lactate accumulation in a concentration dependent manner. The selective inhibition of MUC13 and PKD1 (upstream key proteins of glucose metabolism) that caused the disruption of glucose intake in cancer cells, was observed during steviol treatment. Secondly, steviol interfered with translation-initiation machinery, causing destabilization of the cellular functionality. The impairment of cellular translation process had promoted G1 cell cycle arrest in cells due to the lack of G1-S transition proteins during drug exposure. Collectively, all these events engendered the cellular integrity and directed towards activation of apoptosis. In conclusion, we evaluated the role of MUC13 and PKD1 in aberrant glucose metabolism leading to growth and aggressive metastatic phenotypes of pancreatic cancer. We also demonstrated efficacy of steviol to repress aberrant glucose metabolism via suppression of MUC13 and PKD1 proteins.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0002-6052-9972

DOI

10.21007/etd.cghs.2019.0482

2019-006-Kumari-DOA.pdf (435 kB)
Declaration of Authorship

Available for download on Friday, April 16, 2021

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