Cree, Tabitha (2021) Investigating the role of FK506 binding protein 25 in cell proliferation and differentiation. PhD thesis, Victoria University.

Abstract

Peptidyl prolyl isomerases (PPIase) are a class of enzymes that are required to catalyse the conversion of proline residues from cis to trans conformation. There are several classes of PPIase molecules, including parvulins, cyclophilins, and FK506 binding proteins (FKBPs). Among these PPIase molecules each class contains a conserved PPIase domain that facilitates protein to protein interactions. These PPIase molecules have diverse functions in cellular function and disease progression. FKBPs are a group of immunophilin molecules that are known to interact with immunosuppressant molecules FK506 and rapamycin to stop the immune response and inhibit mTOR, respectively. The structure and function of FKBPs is diverse, these proteins act to facilitate protein to protein interactions, act as co-chaperones, translocate throughout the cell in response to stress events, and bind to DNA. Importantly, FKBPs have been implicated in the pathogenesis of cancer, largely through their roles in co-chaperoning hormone receptors in hormone responsive cancers i.e. breast and prostate cancers. Of particular interest, FKBP25, a 25kDa protein that consists of two functional domains, an N terminal basic helix–loop–helix and C terminal PPIase domain. FKBP25 is known to be involved in protein folding, cytoskeletal dynamics, DNA damage repair, double stranded RNA binding, interacting with the pre-ribosome, and cellular stress responses. Despite the variety of roles that FKBP25 is known to play, there is limited research regarding FKBP25 role in disease and cell differentiation. To address this, initial studies investigated the role of FKBP25 in breast cancer progression and epithelial to mesenchymal transition (EMT). Here it was found that FKBP25 protein expression is reduced in both mesenchymal breast cancer cell types, including BT-549, Hs578t, MDA-MB-231. To further understand the potential role of FKBP25 in breast cancer pathogenesis, a variety of mutations that contribute to malignant transformation were examined. Here it was found that the oncogenic mutations, that are associated with growth pathways in fact increased FKBP25 expression. However, in an epidermal growth factor mediated model EMT in MDA- MB-468 breast cancer cells, it was identified that FKBP25 protein expression was reduced. This implies that the loss of FKBP25 protein expression may be required for de-differentiation and progression of cancer cells. As such, it was hypothesised that FKBP25 protein expression was correlated with the level of cellular differentiation. To examine this hypothesis, next a model of mesenchymal to epithelial transition (MET) was analysed. The C2C12 model of myogenesis to study the role of FKBP25 in an MET-like example of cell differentiation. Previous studies have identified that FKBP25 is the most highly expressed FKBP in skeletal muscle and is expressed in the top 10% of the skeletal muscle proteome. Here it was identified that in proliferative myoblasts there is a higher level of FKBP25 protein expression compared to that of post mitotic myotubes. This was further demonstrated in a model of C2C12 quiescence where it was demonstrated that upon removal from the cell cycle, myoblasts accumulate greater levels of FKBP25 protein expression, which is then reduced upon re-entry to the cell cycle. Interestingly, this trend was not observed in human primary myoblasts, however, was identified in human rhabdomyosarcoma cells which may be due to the presence of p53 and MyoD mutations. Furthermore, in vivo models of muscle plasticity were examined to assess the impact of FKBP25 on skeletal muscle regeneration considering FKBP25 is the most highly expressed FKBP in mature skeletal muscle. Here it was discovered that FKBP25 protein expression is increased in models of regeneration including, chronic mechanical loading, murine muscular dystrophy (mdx), and denervation. It is hypothesised that this was observed due to extensive cytoskeletal remodelling to repair structural damage caused by hypertrophy and atrophy of fibres. Next, we examined the impact of FKBP25 knockdown (25KD) on cell biology and function of MDA-MB-468 and C2C12 cells. 25KD cells were developed using doxycycline inducible SMARTvector (Dharmacon, CO, USA) short hairpin RNA technology. After confirming adequate 25KD, it was observed that in both cell lines 25KD resulted in an increase in proliferation compared to respective non-targeting (NT) cells. Furthermore, in MDA-MB-468 cells, it was observed that there were no changes to invasion outgrowth or migration in vitro. However, it was demonstrated that 25KD resulted in decreased anchorage dependent growth, which could be explained by alterations to cytoskeletal stability. Conversely, in C2C12 myoblasts it was found that 25KD resulted in a significant increase in wound healing migration. Upon investigation of myogenic regulatory factor expression in differentiated 25KD myotubes it was revealed that there were no changes in protein expression. Furthermore, upon measurement of fibre diameter and fusion index it was found that there were no discernible changes to myotube formation. Finally, the influence of 25KD on tubulin regulation and dynamics was assessed. Initially, the presence of microtubule (MT) post-translational modifications was assessed, including detyrosination and acetylation which are associated with MT stability. Both C2C12 and MDA-MB-468 25KD cells showed no changes to stabilising modifications. Similarly, upon examination of MT stabilising protein stathmin, both C2C12 and MDA-MB-468 25KD showed no change to stathmin expression. After this, the impact of 25KD on tubulin polymerisation under control and paclitaxel treated (induction of maximal polymerisation) conditions was explored. However, here no differences in MT polymer content was found in either 25KD in either C2C12 or MDA- MB-468 cells. In conclusion, this thesis has examined the potential role of FKBP25 in cell differentiation and de-differentiation in EMT and MET-like models. It was found that FKBP25 is required for some cell processed including proliferation, anchorage dependent growth, and migration. It was hypothesised that this was a result of cytoskeletal reorganisation and altered MT dynamics, however, this was unable to be demonstrated. Further studies should further examine the impact of 25KD on MT dynamics using methods less prone to error. Nonetheless, FKBP25 was demonstrated to have a role in cell proliferation and differentiation. Maintenance of FKBP25 protein in both cancers and skeletal muscle could help to preserve epithelial-like phenotype and maintain structural integrity, respectively.

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