Thesis Format
Integrated Article
Degree
Doctor of Philosophy
Program
Anatomy and Cell Biology
Supervisor
Penuela, Silvia
Abstract
The skin contains the channel-forming glycoproteins pannexins (PANX) which facilitate cellular communication. PANX1 and PANX3 function in wound repair and keratinocyte differentiation, but unlike PANX1 which is abundant in young skin to establish proper skin architecture, the role of PANX3 in skin homeostasis and aging is unknown. Additionally, PANX1 promotes melanoma carcinogenesis, but little is known about PANX1 and PANX3 in the keratinocytic cancer cutaneous squamous cell carcinoma (cSCC). Here, we used global Panx3 knockout (KO) mice and congenic wildtype controls to investigate PANX3 in the skin. We determined PANX3 was undetectable in wildtype newborn dorsal skin but became upregulated from postnatal day 2-4, where its levels remained consistently high up to 18 months. Panx3 KO mice had thinner skin, a compromised epidermal barrier as well as increased dermatitis incidence and epidermal inflammatory signalling. Furthermore, KO primary keratinocytes showed adhesion deficits most likely due to reduced Wnt signalling. In human cSCC tumours, PANX1 was upregulated but PANX3 mRNA was downregulated compared to patient-matched skin. PANX1 was present in all cSCC tumour regions, where it promoted carcinoma cell growth and migration through its channel activity. Contrarily, using Panx3 KO mice in a chemical carcinoma model, we determined PANX3 reduces the incidence and volumes of pre-cancerous papillomas, highlighting its anti-tumorigenic effects in the skin. Lastly, when surveying human cancer cell lines we discovered a highly abundant ~25 kDa isoform of PANX1 (PANX1-25K) which is increased in cSCC compared to normal skin and promotes cancer cell survival and growth. We determined PANX1-25K lacks the N-terminus of PANX1 and becomes increased with CRISPR/Cas9 ablation of the initial methionine start site of PANX1, but does not result from alternative promotor usage or splicing, suggesting instead that it may be generated through alternative translation initiation. Moreover, PANX1-25K resembles PANX1 biochemically, being glycosylated on novel asparagine residues, phosphorylated and interacting with β-catenin, but exhibits an intracellular localization where it interacts with PANX1. Collectively, in this thesis we identified a new PANX1 isoform and illustrated that PANX1, PANX3 and their isoforms are key regulators of skin homeostasis and aging which become dysregulated in the context of cSCC.
Summary for Lay Audience
The skin helps to regulate body temperature, prevent infection and reduce water loss, and since skin function is tightly linked to its structure, the skin must develop and be maintained properly or it can become cancerous. Pannexins are channel-forming proteins present in the skin that enable cellular communication, but the role of pannexins in skin and skin cancer is unclear. In the first study, we determined the abundance of Pannexin3 in skin was the same in female and male mice and levels increased with aging. We also discovered mice lacking Pannexin3 had thinner skin, with a leaky barrier and increased risk for developing inflammation. In the second study, we obtained patient squamous cell carcinoma tumours (a type of skin cancer arising from the outermost layer of the skin) and normal skin samples from local surgeries to assess pannexin levels and function in this cancer type. We found that more Pannexin1 was present in tumours than healthy skin, whereas Pannexin3 had lower abundance in tumours compared to skin, meaning Pannexin1 and Pannexin3 exhibit an opposite expression pattern. In the same fashion, Pannexin1 promotes the growth and movement of skin cancer cells through its channel activity whereas Pannexin3 reduces the size and incidence of pre-cancerous skin growths. In the third study, we identified a new, shorter form of Pannexin1. This form is present in many different cell types but is more abundant in cancers like squamous cell carcinoma, where it may be critical for cancer cell growth and survival. We suspect this alternative form arises from the same initial cellular instructions used to create the full length Pannexin1 protein but only contains the second half of the protein sequence. This shorter form of Pannexin1 can interact with the full length Pannexin1 inside the cell, and shares many characteristics with the full-length form such as its ability to be modified by sugars and phosphate groups and bind the same interacting partners. Overall, this thesis demonstrates that Pannexin1 and Pannexin3 and their alternate forms are critical in maintaining normal skin health and their abundance and function becomes altered in skin cancer.
Recommended Citation
O'Donnell, Brooke, "The Characterization of Pannexin 1 and Pannexin 3 Channels and their Isoforms in Skin Health and Keratinocytic Cancers" (2024). Electronic Thesis and Dissertation Repository. 10144.
https://ir.lib.uwo.ca/etd/10144
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This work is licensed under a Creative Commons Attribution-No Derivative Works 4.0 License.
Included in
Biochemistry Commons, Cancer Biology Commons, Cell Biology Commons, Integumentary System Commons, Molecular Biology Commons, Skin and Connective Tissue Diseases Commons