Student Projects

  1. Inhibiting organelle contact sites in prostate cancer to impair cell survival

  2. Investigating the role of CaMKK2 in prostate cancer survival

  3. Investigating how phosphorylation puts a break on endocytosis of cell surface receptors

Inhibiting organelle contact sites in prostate cancer to impair cell survival

Prostate cancer (PC) is the second most common cancer in men with 1.4 million new cases diagnosed per year in the world.  The disease is driven by the nuclear hormone transcription factor, androgen receptor (AR). Anti-androgen treatment is a standard of care treatment to slow down the progression of the disease. However, many patients develop resistance to anti-AR treatment (castration resistant PC), while the activity of the AR is maintained. Once it progresses to an advanced lethal disease stage the treatment options are very limited and the median survival is less than 2 years.  To overcome this and provide better outcomes for patients we need to understand the biological mechanisms of the disease better and identify new therapeutic targets.   

Mitochondrial fitness is central to cancer cell survival and meeting the increased energy requirements. This requirement is partly met by interconnection between organelles through membrane contact sites (MCS). In particular, MCS with the endoplasmic reticulum (ER) are critical for regulating cell death, lipid transfer and oxidative stress.   By wrapping around the mitochondria, the ER provides a platform that enhances contact between the organelles, transfer of signalling molecules and lipids, and protects against apoptosis. We recently characterised these contact sites in PC for the first time and demonstrated that there is a positive correlation with histological disease progression to intermediate grade for these dynamic contact sites (Butler & Evergren, 2023). Based on our data a prognostic gene signature was developed that incorporate proteins that promote/maintain/reside in MCS, and we are now looking to validate it. We hypothesise that similar adaptations in MCS occur in castrate-resistant PC and that disruption of these with an anti-MCS drug will sensitise the cells to apoptosis. 

We will establish if membrane contact sites contribute to prostate cancer (PC) and castrate-resistant prostate cancer (CRPC) biology by assessing cell viability and the abundance of contact sites between mitochondria and endoplasmic reticulum (ER) after knockdown or inhibition of target proteins.  

Aims 

  1. Assess whether MCS support castrate-resistant prostate cancer biology.   

  2. Investigate whether the genes in the prognostic MCS signature are essential for mitochondria MCS formation in PC and CRPC cell lines.  

  3. Assess whether a drug that attenuates MCS formation can synergise with Enzalutamide to reduce cell viability. 

Potential impact: Based on our recent publication we hypothesize that membrane contact sites between the ER and mitochondria in PC cells promote cell proliferation and tumorigenesis. This research study will be the first to investigate the role of contacts sites in CRPC, where resistance to standard-of-care treatment develop through molecular adaptations.  Our findings will provide new knowledge on resistance mechanisms and has the potential to identify novel treatment targets that in long-term can be used to improve patient outcomes. 

Techniques employed: Cell culture, shRNA-mediated knockdown, transfections, immunocytochemistry, confocal microscopy, fluorescence assisted cell sorting (FACS), cell viability assays, apoptosis assays, Western blotting. 

Skills/Knowledge required: Basic knowledge in cell biology and statistical analysis.

Investigating the role of CaMKK2 in prostate cancer cell survival

Prostate cancer is the second most common cancer in men with 1.4 million new cases diagnosed per year in the world.  Once it progresses to an advanced lethal disease stage the treatment options are very limited and the median survival is less than 2 years.  To overcome this and provide better outcomes for patients we need to understand the biological mechanisms of the disease better and identify new therapeutic targets. Calcium/calmodulin-dependent protein kinase (CaMKK2) is highly expressed in advanced prostate cancer where it promotes proliferation and tumorigenesis and developing a further understanding of its function is therefore important.   

The Golgi is an essential organelle in the cell that functions both as a sorting hub for proteins and performing essential sugar modifications of proteins.  These sugar modifications, glycosylations, are important for a number of reasons; stabilisations of proteins, intracellular targeting, secretion and protection from proteolytic enzymes. Recent research has demonstrated that glycosylating enzymes are upregulated in prostate cancer and are an important mechanism for promoting cancer cell survival.  These enzymes are distributed across the Golgi into distinct regions of the Golgi stack. This facilitates sequential enzymatic events and promotes the fidelity of protein glycosylation. Newly synthesized and glycosylated proteins are trafficked from the ER to the trans-Golgi where vesicles bud off and deliver these proteins to different organelles or to the plasma membrane. In the face of this constant flux through and from the Golgi there is a requirement for retrograde vesicle trafficking to ensure that Golgi resident enzymes remain in the correct location rather than being lost to the cell surface or other organelles. This is mediated by COPI-coated vesicles. We recently discovered that COPI-mediated vesicle trafficking is regulated by the enzyme CaMKK2, a protein that is highly expressed in advanced prostate cancer (Stewart et al., Cell Death and Disease 2021). We have also observed a deregulation of lipid droplet homeostasis in relation to CaMKK2 expression levels.

Aims

Given that CaMKK2 and glycosylating enzymes are overexpressed in prostate cancer, are regulate by the androgen receptor (a principal driver of prostate cancer) and support tumorigenesis in their own right we hypothesise that they act in concert. Specifically, we hypothesise that (1) CaMKK2 regulates the correct compartmentalisation of glycosylation enzymes in the Golgi and (2) that inhibition or knockdown of CaMKK2 results in mis-localisation of these enzymes to the lysosome or the plasma membrane and promotes cell death (3) CaMKK2 regulates lipid droplet degradation. To test this hypothesis the student will: 

  1. Establish whether the loss of CaMKK2 expression in a prostate cancer cell line alters the cis-, medial-, and trans-Golgi compartmentalisation of established Golgi markers. 

  2. Investigate whether knockdown of CaMKK2 expression results in a re-distribution of three glycosylating enzymes within the Golgi; GALNT2, GALNT7 and ST6GAL1. 

  3. Investigate whether CaMKK2 regulates turnover of lipid droplets and how this supports cell survival.

Techniques employed: Cell culture, transfections, immunocytochemistry, confocal microscopy, fluorescence assisted cell sorting (FACS), cell viability assays, protein assays, Western blotting.