Imagining Human Cancer Progression in a Novel Zebrafish Model

University Of California, San Diego, La Jolla CA

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Abstract

Description¿ Most¿of¿our¿mechanistic¿understanding¿of¿how¿human¿cancer¿cells¿migrate¿and¿invade¿has¿been¿obtained¿by¿ observing¿cell¿behavior¿in¿an¿artificial¿2 D¿environment.¿¿Although¿progress¿has¿been¿made¿using¿this¿ approach,¿important¿new¿evidence¿indicates¿that¿cell¿migration¿in¿2 D¿systems¿does¿not¿completely¿ recapitulate¿events¿associated¿with¿locomotion¿in¿a¿more¿physiological¿environment¿using¿reconstituted¿3 D¿ matrices¿and¿tissue¿explants.¿¿Work¿by¿others¿and¿novel¿evidence¿provided¿in¿this¿research¿proposal¿ demonstrate¿¿invasive¿cells¿can¿utilize¿either¿a¿mesenchymal type¿of¿cell¿invasion¿that¿involves¿formation¿of¿an¿ elongated¿invadapodia¿and¿a¿spindle shaped¿morphology¿or¿a¿primitive¿amoeboid¿¿movement¿that¿involves¿ membrane¿blebbing¿though¿small¿holes¿in¿the¿extracellular¿matrix.¿¿These¿breakthrough¿findings¿prompted¿ the¿hypothesis¿that¿cells¿are¿armed¿with¿different¿invasive¿programs¿that¿allow¿them¿to¿traverse¿complex¿ tissues¿and¿colonize¿foreign¿sites¿in¿the¿body.¿¿Most¿importantly¿though¿these¿findings¿indicate¿that¿ therapeutic¿prevention¿of¿this¿process¿in¿patients¿will¿require¿a¿multifaceted¿approach¿that¿targets¿both¿modes¿ of¿cell¿invasion.¿¿It¿is¿crucial¿then¿that¿we¿identify¿invasive¿mechanisms¿utilized¿by¿disseminating¿tumor¿cells¿ in¿vivo¿so¿that¿the¿appropriate¿therapeutic¿agent(s)¿can¿be¿designed¿to¿completely¿eradicate¿the¿spread¿of¿ cancer¿in¿patients.¿¿However,¿tumor¿cell¿invasion¿is¿a¿complex¿and¿dynamic¿process¿that¿involves¿the¿intricate¿ interplay¿between¿the¿tumor¿cells¿and¿the¿remodeling¿vasculature¿and¿stroma.¿¿Understanding¿this¿process¿in¿ vivo¿has¿been¿difficult¿because¿it¿has¿not¿been¿possible¿to¿visualize¿this¿process¿in¿high¿resolution¿in¿live¿ animals.¿¿To¿address¿this¿problem,¿we¿have¿developed¿a¿novel¿model¿of¿cancer¿progression¿that¿utilizes¿ human¿cancer¿cells¿growing¿in¿optical¿clear¿zebrafish¿genetically¿engineered¿to¿express¿green¿fluorescent¿ protein¿in¿all¿blood¿vessels.¿¿Using¿this¿model¿and¿dual¿color¿high¿resolution¿confocal¿microscopy,¿we¿ discovered¿that¿the¿metastatic¿gene¿RhoC¿induces¿a¿rapid¿cell¿invasion¿process¿that¿facilitates¿cell¿intravasation¿ through¿vascular¿openings¿induced¿by¿VEGF¿secretion.¿¿In¿contrast,¿mesenchymal¿cell¿invasion¿involves¿ formation¿of¿elongated¿invadopodia¿and¿membrane¿integration¿into¿the¿vascular¿wall,¿but¿not¿cell¿ intravasation.¿¿Our¿goal¿in¿the¿proposed¿work¿is¿to¿understand¿the¿signaling¿mechanism¿that¿control¿amoeboid¿ and¿mesenchymal¿invasion¿as¿cells¿intravasate¿and¿how¿the¿vascular¿pores¿form¿in¿response¿to¿VEGF¿ secretion.¿¿Based¿on¿our¿preliminary¿findings¿and¿the¿work¿of¿others,¿we¿hypothesize¿that¿the¿metastatic¿gene¿ RhoC¿mediates¿amoeboid¿invasion¿through¿Rho¿kinase¿activity¿(ROCK)¿and¿myosin¿II mediated¿contractility.¿¿ We¿also¿hypothesize¿that¿PI3K¿harboring¿activating¿mutations¿found¿in¿human¿cancers¿induces¿mesenchymal¿ cell¿invasion¿through¿activation¿of¿the¿FAK Src CAS Crk Rac¿signaling¿module,¿which¿facilitates¿actin mediated¿invadopodial¿protrusion.¿¿We¿hypothesize¿that¿the¿vascular¿pores¿form¿through¿disruption¿of¿cell cell¿junctions,¿which¿is¿regulated¿by¿src¿phosphorylation¿of¿VE cadherin.¿¿Therefore,¿our¿overall¿goal¿is¿to¿ examine¿in¿detail¿how¿RhoC¿and¿mutated¿PI3K¿signaling¿pathways¿regulate¿cancer¿cell¿invasion¿and¿ intravasation¿and¿the¿molecular¿signaling¿mechanisms¿that¿control¿vascular¿pore¿formation.¿¿ ¿¿ Cancer¿cells¿spread¿throughout¿the¿body¿by¿invading¿into¿blood¿vessels¿where¿they¿are¿carried¿to¿ distant¿organs¿and¿form¿secondary¿tumors.¿¿Work¿in¿this¿proposal¿will¿determine¿the¿mechanism¿of¿ how¿ cancer¿ cells¿ invade¿ through¿ the¿ vessel¿ wall¿ utilizing¿ high¿ resolution¿ confocal¿ imaging¿ of¿ optically¿transparent¿zebrafish¿harboring¿metastatic¿human¿cancer¿cells.¿¿¿