Comparative secretome analysis of benign prostate hyperplasia and prostate cancer cell models

Muvirimi, Takudzwa Kevin (2017-12)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Prostate cancer is the second most prevalent non-cutaneous malignancy affecting men in the world. The current method for screening and diagnosis of prostate cancer relies on the measurement of serum kallikrein 3 (KLK-3) in conjunction with the digital rectal exam. However, serum KLK-3 or prostate specific antigen (PSA) as it is better known, is also elevated by benign prostate hyperplasia (BPH). Consequently, a major challenge in the diagnosis of prostate cancer is the ability to discriminate between prostate cancer and BPH. Misdiagnosis of prostate cancer entails unnecessary treatment for patients which results in psychological stress and physical discomfort. Despite the ineffectiveness of PSA as a biomarker, it is still the primary diagnostic tool. This is largely due to lack of an alternative and it is therefore crucial to find a new biomarker which can distinguish malignant prostate cancer from BPH. In this study, the secretome and proteome of four prostate cell lines (BPH-1, LNCaP, PC3 and PNT2) were investigated to identify novel biomarkers for BPH using a MS based proteomic approach. The analysis of the secretome necessitated that the growth conditions of prostate cell lines were optimized to reduce cell death and maximize secreted proteins. Optimal seeding densities were selected following measurements of total protein, LDH and the LDH/protein ratio. Subsequent optimization of protein precipitation methods identified acetone precipitation as the most suitable method for isolating protein from the conditioned media of prostate cell lines. The cell proteome of the four prostate cell lines was comparatively analysed by LC-MS/MS. The total number of proteins identified per cell line were as follows: 2079 in BPH-1, 2081 in LNCaP, 1853 in PC3 and 2137 proteins in the PNT2 cell line. A literature search of the proteins unique to BPH-1 identified mesencephalic astrocyte-derived neurotrophic factor (MANF) as a potential biomarker for BPH. Ingenuity pathway analysis was subsequently used to analyse and identify aberrant pathways in BPH-1. The following pathways were significantly altered in BPH-1: antiproliferative role of transducer of ERB2 in T cell signalling, pyrimidine ribonucleotide de novo biosynthesis, cell signalling and vitamin and mineral metabolism. The optimised secretome samples could not be analysed due to technical difficulties beyond our control. As a proof of concept preliminary analysis of the cell secretome from PC3 and BPH-1 cell lines yielded forty-six and ninety-nine proteins which were unique to each cell line, respectively. The identification of MANF and the antiproliferative role of transducer of ERB2 in T cell signalling during the proteomic analysis both pointed towards an inflammatory response in BPH. Differences in inflammation could therefore be explored in future in order to develop biomarkers unique to BPH and prostate cancer.

AFRIKAANSE OPSOMMING: Prostaatkanker is die tweede mees algemene kanker ter wêreld wat in mans voorkom. Die huidige metode vir die diagnosering van prostaatkanker is afhanklik van serum kallikrein-3(KLK-3)vlakke tesame met ‘n digitale rektumondersoek. Serumvlakke van KLK-3, beter bekend as prostaat-spesifieke-antigeen (PSA), is egter ook verhoog tydens nie-kwaadaardige prostaathiperplasie (BPH). Gevolglik is 'n groot uitdaging in die diagnose van prostaatkanker die vermoë om te onderskei tussen prostaatkanker en BPH. Die misdiagnose van prostaatkanker lei tot onnodige behandelings vir pasiënte wat sielkundige stres en fisiese ongemak tot gevolg kan hê. Ten spyte van die feit dat PSA oneffektief is as ‘n biomerker word dit steeds gebruik as ‘n primêre of hoof diagnostiese hulpmiddel. Die rede hiervoor kan grootliks toegeskryf word aan ‘n tekort aan alternatiewe hulpmiddels en dus om hierdie rede is dit noodsaaklik om ‘n nuwe biomerker te identifiseer wat kan onderskei tussen prostaatkanker en BPH. In hierdie studie is die sekretoom en proteoom van vier sellyne (BPH-1, LNCaP, PC3 en PNT2) ondersoek om nuwe biomerkers te identifiseer vir BPH deur gebruik te maak van ‘n MS gebasseerde proteomiese benadering. Die analiese van die sekretoom het vereis dat die groeikondisies van die prostaatkanker-sellyne geoptimiseer word om seldood te vermy, maar terselfde tyd die maksimum afskeiding van proteiene tot gevolg het. Optimale groeidigthede was geselekteer gevolg deur die bepaling van die totale proteïen, LDH en die LDH/proteïen-verhouding. Die optimisering van proteïenpresipitasie-metodes het asetoonpresipitasie identifiseer as die mees geskikte metode vir die isolering van proteïene vanuit prostaatsellyne se gekondisioneerde groeimedium. Die selproteoom van die vier prostaat-sellyne is in ‘n relatiewe manier geanaliseer deur gebruik te maak van LC-MS/MS. Die totale aantal proteïene wat geidentifiseer is per sellyn was as volg: 2079 in BPH-1, 2081 in LNCaP, 1853 in PC3 en 2137 proteïene in die PNT2 sellyn. ‘n Literatuurstudie van proteïene uniek aan BPH-1 het die mesencefaliese-astrosiet-afgeleide neurotrofe-faktor (MANF) as ‘n potensiële biomerker vir BPH identifiseer. “Ingenuity pathway analysis”-sagteware is gevolglik gebruik om padweë wat afwyk te analiseer en identifiseer. Die volgende padweë is aansienlik verander in BPH-1: die antiprolifererende rol van die “transducer” ERB2 in T-sel seinoordrag, pirimidienribonukleotied de novo biosintese sowel as selsein-, vitamien- en mineral-metabolisme. Die optimiseerde sekretoommonsters kon weens onvoorsiene tegniese probleme nie geanaliseer word nie. Ten spyte hiervan dien die voorlopige analieses van die selsekretoon van PC3 and BPH-1 sellyne as konsepondersteuning en het onderskeidelik ses-en-veertig en nege-en-negentig proteïene opgelewer wat uniek is aan elke sellyn. Die identifikasie van MANF en die antiprolifererende rol van die “transducer” ERB2 in T-sel seinoordrag gedurende die proteomiese analieses is beide ‘n aanduiding van inflammatoriese reaksies in BPH. In toekomstigie studies kan die verskille in inflammasie dus gebruik word om biomerkers uniek aan BPH en prostaatkanker te ondersoek.

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