Role of N-WASP in cell proliferation : implication for organ development and cancer

• Main Author: Verma, Apoorva
• Other Authors: Thirumaran s/o Thanabalu
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/143422
• Institution: Nanyang Technological University

N-WASP regulates actin cytoskeleton and plays key roles in many cellular processes such as cell-proliferation, cell-adhesion, cell-migration and endocytosis which are essential for normal cell physiology. Development of organs such as lungs and mammary glands requires elongation and branching of epithelial tissues which are critically depended on apical-basal polarity, cell adhesion, cell proliferation and interactions with the extracellular matrix proteins. N-WASP has also been shown to regulate cell invasion and EMT (Epithelial to Mesenchymal transition), which are often upregulated during cancer progression. Studies show that N-WASP expression is reduced in breast, colorectal and stage I lung cancer, however its role in lung cancer is not well characterized. It is known from developmental studies that majority of mammary gland development occurs during the adult stage in which extensive branching and duct elongation occurs. Ablation of N-WASP in mice using K5-Cre resulted in significantly decreased number of ducts and sub-ductal branches in the mammary gland. Immunohistochemistry showed significantly reduced number of PCNA positive cells in N-WASP KO mammary glands suggesting reduced proliferation. Moreover, N-WASP KO mammary glands also displayed significantly higher collagen content around the mammary ducts suggesting reduced invasion these ducts. In-vitro experiments with N-WASP knockdown, normal mammary epithelial cell line, MCF10A (MCF10AKD) also showed reduced proliferation, migration, invasion and cell-cell adhesion. Re-expression of N-WASP in MCF10AKD rescued the proliferation defect suggesting N-WASP’s direct role in cell proliferation. Furthermore, MCF10AKD displayed a significantly altered cell cycle profile compared to MCF10Actrl highlighting the role of N-WASP in cell cycle progression. Altogether the experiments show that depletion of N-WASP in mouse mammary glands and in mammary epithelial cell line affect their proliferation, invasion and morphology which ultimately affects the branching and development of mammary gland. Embryonic deletion of N-WASP at E12.5 (Embryonic Day 12.5) in Type II alveolar cells using Sftpc-Cre resulted in hyperplasia with reduced airspaces in the lung. The N-WASP KO lungs also displayed significantly increased number of PCNA positive cells suggesting the role of N-WASP in regulating cell proliferation. While N-WASP deletion alone in the adult stage did not result in any hyper-proliferation phenotype but in KRasG12D induced lung cancer model, N-WASP deletion (N-WASPLKO/KRasG12D) resulted in significantly increased number of tumors in the lung. This suggests that N-WASP negatively regulates cell proliferation and could be a potential tumour suppressor gene. RNA sequencing was carried out to identify potential gene candidates in N-WASPLKO/KRasG12D mice which could be directly or indirectly regulated by N-WASP to enhance lung tumorigenesis. In-vitro experiments with N-WASP knockdown lung cancer cell line, A549 (A549KD) also showed significantly increased cell proliferation in 3D hanging drop model. Screening with a library of kinase inhibitors identified Rho associated kinase (ROCK) inhibitor which significantly and specifically reduced the proliferation of A549KD cells. A549KD cells also showed changes in expression of LIMK and p-cofilin, downstream effectors of ROCK pathway which were restored upon treatment with ROCK inhibitor suggesting that this pathway is probably responsible for increased cell proliferation of A549KD cells. Further studies will be required to elucidate the mechanisms of N-WASP in lung cancer by validating the identified gene candidates and analysing their role in cell proliferation. Overall, the data suggest an important role of N-WASP in lung development and lung cancer.