Author Identifier
Neha Pulyani: https://orcid.org/0000-0002-1421-7847
Date of Award
2025
Document Type
Thesis
Publisher
Edith Cowan University
Degree Name
Doctor of Philosophy
School
School of Medical and Health Sciences
First Supervisor
Elin Gray
Second Supervisor
Aaron Beasley
Third Supervisor
Weitao Lin
Fourth Supervisor
Rodrigo Carlessi
Abstract
Uveal melanoma (UM) is a malignancy arising from the abnormal behaviour of melanocytes present in the middle layer of the eye or uvea. Half of the patients with primary UM eventually develop metastases and 92% of metastatic patients die within 2 years. UM metastasises predominantly to the liver in 95% of cases, with other organ sites including bone and lungs. Therapies such as targeting activated kinases, chemotherapy and immunotherapy have been limited in improving the overall survival of patients, highlighting the need for a better understanding of UM tumours and, particularly, what sustains UM liver metastases.
Preliminary data from our team using single-cell RNA sequencing reveal genes coding for different proteins that are involved in driving interactions between tumour cells and liver cells. In particular, a strong interaction was found between the CD44 receptor expressed on the tumour cells and the extracellular matrix (ECM) ligands such as collagen secreted by hepatic stellate cells (HSCs).
CD44 is a glycoprotein whose structure and function in driving tumour cell proliferation and migration has already been extensively studied in different cancers. However, there is limited understanding of its isoforms and its role in driving tumorigenesis in UM is unknown. Therefore, elucidating the role of CD44 in UM and the signalling pathways activated upon its interaction with the HSCs could reveal novel signalling process in UM and potential therapeutic targets.
The first chapter of the dissertation presents an introduction about UM and comprehensive literature review on CD44 role in cancer. In Chapter 2, the existence of nine splicing variants of CD44 in numerous primary and metastatic UM cells was assessed. The findings revealed that only two CD44 isoforms CD44v2-10 and CD44v3-10 displayed heterogeneous profile, and the remaining isoforms were present across all primary and metastatic UM cell lines.
Chapter 3 describes the impact of CD44 on proliferation and migration of UM cells, along with the transcriptional changes affected by CD44. Here, CD44 Knockouts (KO) were generated using CRISPR approach in two mUM cell lines: OMM1 and OMM1.3. CD44 KOs displayed reduced migration, while decrease in proliferation was observed at 120 hours (hrs) and 96 hrs for OMM1.3 and OMM1, respectively. Bulk RNA sequencing of CD44 KOs revealed alterations in genes related to various hallmark signatures such as epithelial to mesenchymal transition (EMT) and cholesterol homeostasis.
In parallel, we also explored if CD44 could mediate interaction between UM cells and HSCs cells (Chapter 4). Here, we harvested conditioned media (CM) from two HSCs including LX[1]2 and primary HSCs (pHSCs). Both CD44 WT (wild-type) along with their respective KOs were treated with CM to evaluate its potential for proliferation and migration of UM cells. The findings revealed that CD44 KO did not affect proliferation in the presence of pHSCs CM. However, a significant decrease in migration was observed in KOs cells derived from either OMM1.3 or OMM1, when exposed to pHSCs CM. Notably, LX-2 CM enhanced migration and induced profound morphological changes in both WT cells and their CD44 KO counterparts. RNA sequencing of OMM1.3 WT cells cultured with or without LX-2 CM showed reduction in cell cycle-associated genes and profound effect on the expression of genes associated with various pathways and hallmarks such as activation of ECM-receptor interaction and EMT.
Chapter 5 provides a comprehensive discussion on the significance of CD44 in UM in relation to the existing research in carcinomas. It also addresses the study's limitations and suggests future directions for further elucidating the role of CD44 in UM.
DOI
10.25958/xvr3-f223
Access Note
Access to this thesis is embargoed until 2nd September 2027
Recommended Citation
Pulyani, N. (2025). Investigating splicing variants and functional role of CD44 in uveal melanoma. Edith Cowan University. https://doi.org/10.25958/xvr3-f223