The Translational Application And Mechanism Of Albumin Uptake Via Macropinocytosis In Thyroid Cancer

Background: Macropinocytosis is defined as the non-specific endocytic uptake of extracellular material by cells in a clathrin-independent manner which could fuel tumor to sustain their survival and growth.Human serum albumin is a water soluble and the most abundant protein in the plasma which exhibits extended circulating half-life,and its properties have long been exploited for diagnostics and therapies.Albumin is the substrate of extracellular matrix and could enter into cancer cells through macropinocytosis to supply nutrient needs for cancer cells.Thyroid cancers are the most common endocrine malignancy and classified as differentiated thyroid cancer(papillary thyroid cancer(PTC)and follicular thyroid cancer(FTC)),medullary thyroid cancer and anaplastic thyroid cancer(ATC)according to the histology.BRAFV600 E is the most frequently oncogene mutation in thyroid cancers,especially in papillary thyroid cancer,and is associated with aggressiveness of this type of tumor.Macropinocytosis is recognized as a prominent scavenging pathway in cancers with oncogenic mutations that drive constitutive RAS or PTEN mutation,e.g.,in pancreas cancer,lung cancer and prostate cancer.However,little is known of macropinocytosis and albumin uptake in FTC,PTC,and ATC despite BRAFV600 E mutation in these malignancies.Therefore,the experiments in this thesis will help us to investigate the role of macropinocytosis in thyroid cancer,and underly the determined factors of albumin uptake.Taken all together to enhance our understanding of thyroid cancer,and to provide theoretical basis for exploring potential more effective treatments for aggressive thyroid cancer.Objective: To investigate the constitutive macropinocytosis in thyroid cancer,the correlation between albumin uptake and macropinocytosis,and measure the effects of albumin-conjugated drugs in thyroid cancer;To determine different oncogenic mutations,especially BRAF/MEK/ERK signaling pathways,on albumin uptake in thyroid cancer;to explore the effect of metabolism-related signaling pathways on albumin uptake in ATC;and the tumor-associated macrophages influence ATC.Methods: We assessed cellular uptake of dextran and serum albumin was quantified across n≥246 individual cells using fluorescence microscopy in triplicate experiments to identify macropinocytosis in thyroid cancer,and analyze the subcellular co-localization of dextran and albumin,macropinocytosis inhibitor effects on albumin uptake to identify the relationship between albumin uptake and macropinocytosis.The in vivo uptake of serum albumin was assessed across n ≥ 3 replicates of an immunocompetent orthotopic model of BrafV600 E p53-/-ATC in B6129SF1/J mice.The same mouse model was used to measure the effects of an albumin-drug conjugate based on the microtubuledestabilizing monomethyl auristatin E(MMAE)linked to serum albumin via a cathepsin-cleavable peptide(Alb-vc-MMAE)on tumor volume using n ≥ 10 tumors per group.Thyroid follicular cells were transfected with ectopic BRAFV600 E or KRASG12 D plasmid,and PTEN si RNA,p-ERK levels were monitored by immunofluorescence,and thyroid cancer cells were treated with MAPK inhibitors,RET inhibitors,and PI3Kαinhibitors to determine these molecule effects on macropinocytosis and its mediated albumin uptake.MAPK inhibitors dabrafenib and trametinib were treated to ERK triple reporter thyroid cancer cells and the tumor-bearing mice,the correlation between ERK and albumin uptake was investigated by fluorescence or confocal microscopy on cells or tissue,and the effect of MAPK inhibitors on cellular component in thyroid cancer was analyzed via flow cytometry.Potential targets to further promote ATC cells albumin uptake were selected by bioinformatic analysis and then through a panel of multiple targeted small molecule drugs,and using Western blot to analyze the signaling pathways response to si RNA IGF1 R or IGF1 R inhibitors,and additional IGF1,the published single cell sequencing data was analyzed to determine the the IGF1 and IGF1 R distribution.Bone marrowderived macrophages(BMDMs)were co-cultured with murine ATC cells at last to explore the macrophages effects on ATC albumin uptake.Results: FTC,ATC and partial PTC exhibited constitutive macropinocytosis,fluorescence microscopy showed subcellular albumin co-localized with dextran,macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl)amiloride(EIPA)reduced cellular albumin uptake by roughly 50%,consistent with macropinocytosis.ATC tumors accumulated serum albumin up to 8.8% ID/g(injected dose percentage per gram),and Alb-vc-MMAE,but not MMAE alone,blocked tumor growth by >90%.Ectopic BRAFV600 E mutation enhanced follicular thyroid cells macropinocytosis and albumin uptake by 2-fold,albumin uptake is related to ERK activity,MAPK inhibitors trametinib and dabrafenib selectively reduced ATC cells albumin uptake by 50% while has little effect on tumor associated macrophages(TAMs).Despite BRAF/MEK/ERK signaling pathway,PTEN loss and PI3 k signaling modulated macropinocytosis and albumin uptake in thyroid cancer.ATC albumin uptake increased by up to 230% with metformin or insulin like growth factor 1 receptor(IGF1R)inhibitors in monoculture but not in vivo.Inhibition of IGF1 R activated AMPK signaling pathway,and additional IGF1 blocked increased albumin uptake effect by inhibition of IGF1 R.IGF1R mainly expressed on ATC cells while IGF1 expression matched with C1QC+ macrophages.BMDMs diminished inhibition of IGF1 R effects on ATC cellular albumin uptake in co-culture.Conclusion: The presence of constitutive macropinocytosis in thyroid cancer cells mediates the albumin uptake in thyroid cancer cells;large amounts of albumin accumulated in ATC tissue,and albumin-binding drug effectively inhibits the growth of ATC in mice;BRAFV600E mutation enhances the macropinocytic albumin uptake in thyroid cells;macropinocytic albumin uptake is dependent on the activity of the BRAF/MEK/ERK signaling pathway;Inhibition of IGF1 R and activation of AMPK enhance albumin uptake in ATC cells to some extent,but the presence of additional IGF1 and macrophages blocked this phenomenon.