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Mass Spectrometry-Based Chemoproteomic Profiling of Nav1.7, a Voltage-Gated Sodium Channel

Periodic Reporting for period 1 - MaSCheNav (Mass Spectrometry-Based Chemoproteomic Profiling of Nav1.7, a Voltage-Gated Sodium Channel)

Reporting period: 2015-11-01 to 2017-10-31

(The project described in this report has been subjected to an amendment, approved by the EU commission, due to the unexpected closure of the industrial research unit involved as a partner organisation in the original application.)

The project focussed on the interaction between protein lipidation and cancer, looking in particular at the role of cancer/stroma communication in a hetero-cellular environment. Cancer is known for having an altered lipid metabolism, due to cancer cells’ markedly increased need for lipids, to support their growth. Moreover, de novo lipogenesis occurs downstream of various common oncogenic events. Protein lipidation is a unique modification enabling protein interaction with membranes. This modification is thus involved in vesicular trafficking and signalling and is crucial for cellular communication, which plays a major role in cancer. In particular, cancer-stroma hetero-cellular signalling is fundamental in supporting tumour growth. The stroma secretes growth factors and cytokines and deposit extracellular matrix, leading to an increased growth of fibrous tissue. The latter, in turn, implies reduced perfusion, thus potentially contributing to therapeutic resistance in many cancers, and constituting an interesting pharmacological target for combination therapy.
Therefore, the altered lipid scenario in cancer could affect protein lipidation, which in turn plays a crucial role in many signalling events driving cellular communication in the tumour microenvironment. Understanding this complex interplay will help us advance in the development of novel therapeutics to tackle cancer in a more comprehensive way.
In the context of prostate cancer, the androgen receptor has been shown to promote cancer growth through its activity as a transcription factor, i.e. androgens activate it and thus induce the expression of a series of target proteins. I have looked at the effects of androgens on a model cell line for prostate cancer through mass-based proteomics analysis. The results showed a significant over-expression, upon androgen treatment, of several proteins involved in lipid metabolism, thus proving an existing correlation between the two phenomena. Subsequently, I have studied in depth the lipidated proteome in such a context, using a set of tools (probes and capture reagents) developed in the host laboratory. In particular, I have found several significant hits for protein palmitoylation, a very common lipid modification that enables protein interaction with cellular membranes. The expression of none of these proteins is androgen-regulated, suggesting that androgens are actually affecting their plamitoylation levels, possibly through the regulation of lipid metabolism and downstream events related to lipids availability.
I have also looked at heterocellular communication in the context of Pancreatic Ductal Adenocarcinoma (PDAC). I have studied PDAC interaction with the stroma by means of cell type-specific mass-based proteomics in co-culture, a cutting-edge technique in chemical biology. This technique enables to work in direct co-cultures, thus guaranteeing a more efficient intercellular signalling and constituting a better mimic of the tumour microenvironment with respect to the previous state-of-the-art systems. I have carried out this study with innovative transgenic mouse-derived cancer cells. These cells are characterized by a pancreas-specific, doxycycline-inducible, expression of mutant Kras. Kras represents one of the most frequently activated oncogenic drivers in cancer (>90% of PDAC tumours). Mutant Kras oncogenic effects have also been reported to happen in a non-cell-autonomous way, through Sonic hedgehog (Shh) mediated signalling to the stroma. Shh signalling has been shown to enhance the growth of fibrous tissue, which in turn leads to reduced perfusion, thus potentially contributing to therapeutic resistance in many cancers.
A number of proteins resulted to be significantly up- or down- regulated in co-culture with respect to the mono-culture for both cancer and stroma cells. An enrichment analysis showed that proteins that are overexpressed in the stroma in co-culture are associated with the cell periphery, cell ruffles and extracellular vesicles, indicating that reciprocal communication is activated by the presence of the cancer cells. An interesting hit was Chloride intracellular channel protein 1 (Clic1), which was upregulated in co-culture for the stroma cells. Clic1 has been implicated in multiple cancers and has been found to be clinically overexpressed in pancreatic cancer tissues and in glioblastoma, where it is secreted via extracellular vesicles and it seems to have a role in the tumour proliferative capacity. Clic3, another member of the same protein family, has been recently shown to be secreted by the stroma in breast cancer where it drives cell invasion. We can therefore speculate that Clic1 overexpression is induced by the cancer cells in order to favour their growth.
It is well known that Shh needs to be palmitoylated before its secretion from cancer cells, to act on the receiving cell membrane receptor and activate its signalling cascade. Therefore, I was interested in looking at the effects of the inhibition of Shh palmitoyl transferase, Hhat. I successfully verified doxycycline-induced Kras activation, to confirm the oncogenic state of the model PDAC system. However, several analytical techniques to evaluate Shh levels led to the conclusion that these cells express only low amounts of this signalling protein. Therefore, I have translated this technique to a human pancreatic cancer system expressing high levels of Shh, with high physiological relevance. This additional model system, developed for the first time, will constitute the base for the work of a follow-up research project in the host laboratory, building on the results achieved during the duration of the fellowship.
With this project, I have combined several techniques lying at the cutting-edge of chemical biology and proteomics, to study the relation between protein lipidation and cancer in an unprecedented way. I have proven a correlation between prostate cancer activation by androgens and lipid metabolism. Moreover, I have shown how androgens significantly affect palmitoylation levels of several proteins, thus strongly suggesting a correlation between cancer regulators and protein lipidation. Furthermore, I have studied hetero-cellular communication in a pancreatic ductal adenocarcinoma/stroma model. I have showed the overexpression, in stroma cells in co-culture, of proteins associated with signalling processes and the interaction with the extracellular matrix. Moreover, Shh signalling has been suggested to be involved in therapeutic resistance in this cancer and there is an extreme urge to investigate this more in depth. To this aim, I have developed the first human pancreatic cancer model for cell type-specific mass-based proteomics in co-culture, which will be subject to further studies. Understanding this complex interplay represents a crucial step towards a more comprehensive therapeutic approach for cancer, focussing on the interaction between the different players in the tumour microenvironment.