Date of Award

5-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmaceutics

Research Advisor

Subhash Chauhan, PhD

Committee

Mahul Amin, MD Stephen Behrman, MD Santosh Kumar, PhD Yi Lu, PhD

Abstract

Colorectal cancer (CRC) is one of the most prevalent cancer worldwide with a 5% lifetime incidence in developed countries. It is third most common cause of cancer related death in the United States and the second deadliest when men and women are combined. Encouragingly due to changes in dietary lifestyle, screening colonoscopy, and advancement in treatments the mortality has decreased in recent years. Most sporadic CRCs develop from polyploid adenomas and are preceded by intramucosal carcinomas (stage 0), which can progress into more malignant forms. This developmental process is known as the adenoma-carcinoma sequence. Early detection and endoscopic removal are crucial for CRC management. The overall 5-year survival of CRC jumps noticeably from 66% to 91% if it can be diagnosed and treated at early stages but drops down to 14% when the disease has metastasized. Metastasis is the cause of 90% of all cancer related deaths, which necessitates the need to understand the mechanisms regulating different progression steps. Metastasis is a complex and multistep process, whereby cancer cells leave the primary tumor and colonize in new tissues. However, only about 0.02% of tumor cells that detach from the primary tumor are successful in forming a metastatic lesion. After intravasation, anchorage independent survival (Anoikis resistance) of primary tumor cell is one of the key steps in metastasis.

The mucin MUC13 when aberrantly overexpressed in cancer has been found to lead to poor outcomes in Pancreatic, Ovarian, Liver, and Colorectal cancer. When overexpressed in these cancers MUC13 has been shown to increase the oncogenic activity and survival of cancer cells. The oncogenic nature of MUC13 indicates a possible role in the development of Anoikis resistance and CRC metastasis.

To understand the mechanism behind Anoikis resistance we developed and optimized an Anoikis induction model using the low adhesion poly hema coated cell culture dishes. Two isogenic cell lines SW480 (primary tumor site) and SW620 (metastatic tumor site) were used for this study. In our initial experiments SW620 demonstrated less cell death compared to SW480 during Anoikis induction. High MUC13 expression was observed in SW620 between 24-36 hours compared at 0 hours. SW620 further showed an increase in the expression of anti-apoptotic protein Bcl2 also. Overexpression of MUC13 in SW480 (SW480+MUC13) cells increased oncogenic phenotype such as invasion, migration, proliferation, and colony size as compared to SW480+Vector control cells. Downregulation of MUC13 in SW620 cells resulted in decreased oncogenic traits.

The role of MUC13 in Anoikis resistance was further investigated in the overexpression and knockdown cell lines. When overexpressing MUC13 in SW480 cells we found increased survival compared to SW480+Vector cells. Bcl2 expression increased while Cleaved-Caspase 3 showed decreased expression in SW480+MUC13 compared to SW480+Vector cells. Increased MUC13 expression also increased the metastatic potential of SW480 cells in mice. After 36 hours of Anoikis induction and then injection into mice through the tail vein, MUC13 mice had an increased tumor burden compared to Vector mice, with METs forming in the liver, kidneys, and lungs. This would indicate that MUC13 plays a larger part in CRC metastasis and specific in the development of Anoikis resistance.

To understand the mechanism involving MUC13 in Anoikis resistance, we studied kinases after Anoikis induction in the cell lines, followed by quantitative proteomics. We were able to find changes in transcription factor YAP1 and YAP1 and β-catenin, which are known to play vital roles in cellular development. In recent studies YAP1 and β-catenin have been shown to form a complex that promotes tumor cell survival and increased tumorigenesis. High MUC13 expression between 24-36hrs was observed, leading to an increase in nuclear localization of the known survival complex YAP1/β-catenin. Further in-vitro analysis indicated that MUC13 plays a vital role through direct cooperation with YAP1 and β-catenin, and together with increased nuclear localization of YAP1/MUC13 and β-catenin/MUC13 complexes, results in increase expression of pro-survival genes. This relationship was further examined and validated in an in-vivo mouse model, in which high MUC13 expression led to increased YAP1 and β-catenin expression and tumorigenesis. A correlation was further observed between MUC13 and YAP1 expression in human CRC patient tissue samples with high expression in tumor tissues compared to NAT and increased nuclear localization with elevated expression for both MUC13 and YAP1. The novel interaction between YAP1 and MUC13 defines a new mechanism in which cells develop Anoikis resistance in CRC.

Genetic variations in genes are a well-known aspect of most diseases. This is especially true for cancer. Genetic variations in mucins such as MUC1 and MUC5AC have been found to increase the risk of stomach cancer and certain Allele mutations nearly doubling that risk in. We investigated if MUC13 had any genetic variants and found 5 different splice variants. Three of those splice variants were found to be non-coding. We then investigated the two protein coding variants consisted of a 512aa (Long form) and 187aa (Short form). We found that MUC13-LF was responsible for generating the oncogenic phenotype associated with adherent MUC13 expression. MUC13-SF however, showed a decrease in migration and invasion when overexpressed in MUC13 null cell lines.

These studies suggest an oncogenic function of MUC13 in CRC via influencing multiple signaling pathways however, its role in cancer metastasis is remains elusive. In this study, we discovered how MUC13 facilitates metastasis after dissemination of tumor cells from the primary tumor site through influencing interaction and nuclear translocation of YAP1 and β-catenin followed by the expression of their downstream pro-survival and metastasis genes. After escaping from the primary tumor this MUC13 driven molecular mechanism provides a crucial survival advantage to anchorage independent circulating tumor cells, leading to successful and extravasation and homing new distant site for cancer metastasis. The interaction between YAP1 and MUC13 provides a new therapeutic in the prevention of metastasis that in combination with current chemotherapy could limit the tumor to its primary site.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0002-2019-9411

DOI

10.21007/etd.cghs.2021.0533

2021-006-Doxtater-DOA.pdf (378 kB)
Declaration of Authorship

Available for download on Tuesday, May 10, 2022

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