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To date, the field of protein phosphorylation has focussed largely on cytoplasmic and nuclear protein kinases and the roles they play in health and disease. The field of protein phosphorylation started in 1883 when the secreted milk protein casein was found to contain phosphorus (1). Since then multiple extracellular proteins like egg-yolk phosvitin, fibrinogen, cytokines, collagen, hormones, insulin-like growth factor binding proteins (IGFBPs), matrix-metalloproteases, and aggrecanases have been reported to be (often heavily) phosphorylated (2). However, the functions of these extracellular phosphorylation events have not been established. It was not until 2012 that an atypical secretory pathway kinase, Fam20C, was identified which phosphorylated the majority of these extracellular proteins on a canonical S-x-E/pS motif (3) and is responsible for generating ~75% of human serum (4) and cerebrospinal fluid phosphoproteins (5). Fam20C has over 100 substrates in the extracellular space and no other kinases phosphorylate pS-x-E motifs in the secretory pathway (6). Interestingly, many of these established Fam20C substrates are remarkably overexpressed in glioma tumours and secreted into the blood of grade IV glioma or glioblastoma multiforme (GBM) patients (communications from other scientists), suggesting a potential link to major mechanistic inputs in disease progression. Out of ~7 million brain cancer patients worldwide, GBM has the worst patient outcome with no known biomarkers for early detection or risk stratification for patients. About 88% of GBM tumours are isocitrate dehydrogenase 1 (IDH1) wild-type which correlates with highly heterogeneous and incurable (<5% 5-year survival) disease progression (7, 8). Furthermore, almost all IDH1 wild-type GBM cases exhibit polyploidy of whole chromosome 7 (9) leading to overexpression of resident oncogenic protein kinases EGFR, c-MET, and CDK6. Hence, hypothetically, overexpression of Fam20C in GBM would lead to stoichiometrically higher phosphorylation of the secreted substrates. Phosphoproteomics across various different cancer cells with/without Fam20C depletion identified the top 3 overlapping substrates to be OPN, IGFBP7, and interleukin6 (IL6), proteins whose roles in glioblastoma progression are well-established (10-12), but the roles of their phosphorylation remain unknown. Fam20C depleted cells exhibit dramatically reduced proliferation and invasion phenotypes which can be rescued by supplementing the Fam20C-depleted cells with parental conditioned media suggesting that the extracellular phosphoproteome regulates Fam20C-mediated cancer invasiveness (6). Thus, it is essential to investigate how Fam20C promotes GBM progression and which substrates of Fam20C are the prime oncogenic drivers of GBM.
Fam20C is resident on chromosome 7, which exhibits glioma-specific polyploidy during early initiation stages often 2-7 years prior to diagnosis (13, 14). Consequently, Fam20C is markedly overexpressed in the most invasive stage IV IDH1 wild-type glioma (Fig 1A-C). Indeed, mice challenged with Fam20C-depleted orthotopic glioma survive remarkably better than those bearing parental glioma cells (Fig 1D). Primary glioma cells lacking Fam20C exhibit slower proliferation (Fig 1E) while breast cancer cells depleted of Fam20C exhibits slower matrigel-invasion (Fig 1F) and slower migration which can be rescued by adding conditioned media of parental cells (Fig 1G). This suggests that extracellular substrates modified by Fam20C promote the pro-migratory phenotype of the cancer cells. On a similar note, mice with a brain-specific Fam20C knock-out survive orthotopic glioma challenge much better than floxed counterparts (Fig 1H) suggesting that Fam20C not only drives primary glioma proliferation but also provides stromal support to the tumour as well.
Specific Aims: I hypothesize that Fam20C promotes GBM via phosphorylation of IGFBP7, IL6, and OPN. Other, as yet unidentified substrates, might also play a role. I propose the following aims:
Aim 1: Establish the intracellular signalling cascades engaged by Fam20C substrates
Aim 2: Elucidate the role of Fam20C substrates in promoting GBM invasiveness
Aim 3: Identify novel Fam20C substrates promoting GBM in vivo
Successful completion of the aims will directly impact ~7 million brain cancer patients, especially those with stage IV GBM, for whom the median survival is a dismal 12-15 months from initial diagnosis. My proposal could not only establish Fam20C as a therapeutic target, but the plethora of extracellular substrates could provide us with multiple early detection/prognostic/stratification biomarkers in brain cancer.
Fig 1 Fam20C is a glioma biomarker. (A) Higher Fam20C mRNA expression correlates with poor overall patient survival in LGG (low grade glioma) and GBM. (B) Fam20C expression is higher in IDH1 WT and (C) WHO grade IV glioma. (D) PDX GBM184 Fam20C KO bearing mice survive better upon intracranial implantation in nude mice. (E) Proliferation assay for PDX line GBM184 WT vs Fam20C KO and GBM242 scrambled control vs shFam20C knockdown. (F) Human breast cancer cells with depleted Fam20C fails to invade in a 3D Matrigel invasion assay. (G) The reduced proliferative phenotype of Fam20C depleted breast cancer cells can be rescued with parental cell conditioned media only (H) Brain-specific Fam20C depletion allows better survival for mice allografted with GL261 cells.
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