The role of Serum Amyloid A in NLRP3 inflammasome signalling in breast and colon cancer

Thesis (PhD)--Stellenbosch University, 2022.ENGLISH ABSTRACT: Introduction: Cancer is a complex disease with multiple interactions targeting the organism on cellular, tissue and systemic levels. The main research focus for the past decades has been on the genome and on the molecular level where signaling pathways were dissected for the development of targeted therapies. However, in order to develop more efficient therapeutic regimes, a better understanding on systemic level is required. Over the past few years, the role of serum amyloid A (SAA) has gathered significant evidence which highlights its role in the pathogenesis of several cancers, including breast and colorectum carcinomas. To date, SAA has been shown to bind to several pattern recognition receptors, which might suggest that inflammasomes play a role in the tumour-promoting properties of SAA. Inflammasomes are cytoplasmic multiprotein complexes characterized by a sensor protein, an adaptor protein, and inflammatory caspases. However, the role of inflammasomes in cancer remain controversial. The aim of this study was therefore to investigate the role of SAA in inflammasome signaling in breast and colon cancer. Methods: In this 3-part animal study, tissues were subjected to immunoblotting, real-time PCR, haematoxylin and eosin staining and immunohistochemistry. For the first inflammatory model, wild-type and SAA double knockout C57BL/6 mice received 2.5% dextran sulfate sodium, which was administered for a total of 5 days. To assess tumourigenesis, colitis-associated cancer and triple negative breast cancer models were used, respectively. For colitis- associated cancer, wild-type and SAA double knockout C57BL/6 mice received an intraperitoneal injection of 12.5 mg/kg azoxymethane. After one week, dextran sulfate sodium treatment was administered at a concentration of 2.5% for a total of 5 days, followed by a recovery period of 16 days. Dextran sulfate sodium treatment was administered for a total of 3 cycles. Triple negative breast tumours were established in wild-type and SAA double knockout C57BL/6 mice by injecting EO771 cells subcutaneously at the fourth mammary fat pad. The experimental endpoint was reached when tumour volumes reached 300-400 mm3. Results: In this study we have showed that in an in vivo model of dextran sulfate sodium induced colitis, SAA ablation exerted pro-inflammatory properties independent of the NLRP3 inflammasome. The ablation of serum amyloid A1/2 was associated with the increased expression of pro- inflammatory cytokines. In contrast, in an in vivo colitis-associated cancer and in a triple negative breast cancer model, the ablation of SAA suppressed canonical NLRP3 inflammasome activation, which was associated with anti-inflammatory properties. These findings suggest that during tumourigenesis, SAA functions as an endogenous damage associated molecular pattern in the tumour microenvironment. Conclusion: Here we show for the first time, in models of CAC and TNBC, the novel role of SAA in the activation of the NLRP3 inflammasome and the generation of pro-inflammatory cytokines, two mechanisms known to promote tumour development and metastasis. This study emphasizes the notion that the tumour-induced systemic environment acts as a critical regulator of cancer progression and metastasis. In conclusion, simultaneously targeting SAA and NLRP3 components could be beneficial for cancer treatments.AFRIKAANSE OPSOMMING: Inleiding: Kanker is 'n komplekse siekte met verskeie interaksies wat die organisme op sellulêre, weefsel- en sistemiese vlakke teiken. Die hoof navorsingsdoel vir die afgelope dekades was op die genoom sowel as seinoordragpaaie op molekulêre vlak te dissekteer vir die ontwikkeling van geteikende terapieë. Om meer doeltreffende terapeutiese prosedures te ontwikkel, is 'n beter begrip op sistemiese vlak egter nodig. Oor die afgelope paar jaar het die rol van serum amiloïed A (SAA), 'n akuut-fase proteïen, hoofsaaklik deur hepatosiete geproduseer, beduidende bewyse gelewer vir die rol daarvan in die patogenese van verskeie kankers, insluitend bors- en kolorektumkarsinome. Dit is al bewys dat SAA aan verskeie reseptore bind, soos TLRs, wat voorstel dat inflammasome 'n rol speel in tumorgenese. Inflammasome is sitoplasmiese multiproteïen-komplekse wat gekenmerk word deur 'n sensorproteïen, 'n adapterproteïen en inflammatoriese kaspases. Die rol van inflammasome in kanker bly egter omstrede. Die doel van hierdie studie was dus om die rol van SAA in inflammasoom aktivering in bors- en kolonkanker te ondersoek. Metodes: In hierdie 3-delige dierestudie is weefsels ondersoek deur gebruik te maak van qPCR, hematoksilien- en eosienkleuring en immunohistochemie. Vir die eerste inflammatoriese model het wilde-tipe (WT) asook muise wat nie SAA gene besit nie (SAADKO), 2.5% dekstraansulfaatnatrium (DSS) ontvang, wat vir 'n totaal van 5 dae toegedien is. Om tumorgenese te ondersoek, is kolitis-geassosieerde kanker- (CAC) en trippel negatiewe borskankermodelle (TNBC) onderskeidelik gebruik. Vir CAC het wilde-tipe en SAADKO C57BL/6 muise 'n intraperitoneale inspuiting van 12.5 mg/kg asoksimetaan ontvang. Na een week is DSS-behandeling toegedien in geoutoklaveerde drinkwater teen 'n konsentrasie van 2.5 % vir 'n totaal van 5 dae, gevolg deur 'n herstelperiode van 16 dae. DSS-behandeling is vir 'n totaal van 3 siklusse toegedien. TNBC gewasse is gevestig in WT en SAADKO C57BL/6 muise deur EO771 selle onderhuids in die vetweefsel van die vierde melkklier in te spuit. Die eksperimentele eindpunt is bereik toe tumorvolumes 300-400 mm3 bereik het. Resultate: In hierdie studie het ons getoon dat in 'n in vivo model van dekstraansulfaat natrium- geïnduseerde kolitis, SAA ablasie pro-inflammatoriese eienskappe uitgeoefen het onafhanklik van die NLRP3 inflammasoom. Die ablasie van SAA1/2 was geassosieer met die verhoogde uitdrukking van pro-inflammatoriese sitokiene. In teenstelling hiermee, in 'n in vivo kolitis- geassosieerde kanker en in 'n trippel negatiewe borskankermodel, het die ablasie van SAA kanonieke NLRP3-inflammasoomaktivering onderdruk, wat geassosieer was met anti- inflammatoriese eienskappe. Hierdie bevindinge dui daarop dat SAA as 'n endogene skade- geassosieerde molekulêre patroon in die tumor mikro-omgewing tydens tumorgenese, funksioneer. Gevolgtrekking: Hier wys ons vir die eerste keer, in modelle van CAC en TNBC, die nuwe rol van SAA in die aktivering van die NLRP-inflammasoom en die generering van pro-inflammatoriese sitokiene, twee meganismes wat bekend is om tumorontwikkeling en metastase te bevorder. Hierdie studie beklemtoon die idee dat die tumor-geïnduseerde sistemiese omgewing optree as 'n belangrike reguleerder van kankerprogressie en metastase. Ten slotte, deur gelyktydig SAA en NLRP3-komponente te teiken deur reseptore te inhibeer of deur stroomafwaartse seinoordrag meganismes te voorkom, kan dit voordelig wees vir kankerbehandelings.Doctora