Title Karakterizacija globalnih proteomskih profila stanica raka debelog crijeva čovjeka u ovisnosti o statusu gena BRAF Title (english) Characterization of global proteome profiles of human colon cancer cells depending on the status of the BRAF gene Author Veronika Horvat Mentor Mirela Sedić (mentor)Mentor Marko Klobučar (mentor)Committee member Nicholas Bradshaw (predsjednik povjerenstva)Committee member Krešimir Pavelić (član povjerenstva)Committee member Mirela Sedić (član povjerenstva)Committee member Marko Klobučar (član povjerenstva)Granter University of Rijeka Defense date and country 2018-12-14, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract Protein BRAF je signalna serin/treonin protein kinaza uključena u
aktivaciju MAPK signalnog puta koji utječe na staničnu diobu, rast,
diferencijaciju i preživljenje. Mutacije BRAF, kao što je BRAF V600E,
dodatno aktiviraju MAPK signalni put što pojačava proliferaciju i preživljenje
tumora. BRAF V600E mutirani kolorektalni karcinom predstavlja podskup
kolorektalnog karcinoma karakteriziran lošim cjelokupnim preživljenjem i
ograničenim odgovorom na kemoterapiju i ciljanu terapiju. Unatoč brojnim
studijama koje ukazuju na pozitivan odgovor pacijenata oboljelih od
kolorektalnog karcinoma s BRAF mutacijom na kombiniranu inhibiciju
BRAF/EGFR, BRAF/MEK i BRAF/EGFR/MEK, ukupna stopa odgovora i
medijan preživljenja bez progresije nisu značajni, što dodatno naglašava
potrebu za učinkovitijim terapijama za ovu specifičnu populaciju pacijenata.
Stoga je svrha ovog diplomskog rada proširiti postojeće znanje o
signalnim putevima i staničnim procesima koje regulira BRAF V600E
mutacija u stanicama kolorektalnog karcinoma čovjeka. U tu svrhu,
provedeno je globalno proteomsko profiliranje u rasponima pH 3-10 NL i 4-
7 staničnih linija kolorektalnog karcinoma s divljim tipom BRAF (HTC116,
SW620) i BRAF V600E mutacijom (RKO, HT29) uporabom
dvodimenzionalne poliakrilamidne gel elektroforeze i MALDI-TOF/TOF
masene spektrometrije. Pri tome smo identificirali dvadeset proteina sa
statistički značajnom (p0.05) promjenom razine ekspresije u BRAF V600E
mutiranim u odnosu na divlji tip BRAF stanica kolorektalnog karcinoma.
Većina identificiranih proteina uključena je u stanične procese koji su važni
za progresiju tumora uključujući staničnu komunikaciju, stanični ciklus i
rast.
U ovom istraživanju otkriveno je nekoliko novih proteina biomarkera
potencijalno povezanih s agresivnim fenotipom BRAF-mutiranog
kolorektalnog karcinoma. Ti proteini uključuju proteine s potencijalnom
onkogenom ulogom, kao što je protein 5 koji sadrži MORN ponavljanje,
protein cinkovog prsta 230, eukariotski elongacijski faktor 2 kinaza i
serin/treonin protein kinaza 40, kao i one koje mogu djelovati kao tumorski
supresori, uključujući stanični receptor virusa hepatitisa A2 i Ras-povezan
protein Rab-40A. Potrebne su daljnje imunohistokemijske analize kako bi
se utvrdila uloga ovih proteina u patogenezi BRAF V600E-mutiranog
kolorektalnog karcinoma i procijenila njihova prognostička relevantnost.
Specifično ciljanje eukariotskog elongacijskog faktora 2 kinaze i
serin/treonin protein kinaze 40 se može razviti kao nova strategija za
kontrolu BRAF mutiranih kolorektalnih stanica i povećanje njihove
osjetljivosti na kemoterapijske lijekove.
Abstract (english) The BRAF protein is a signalling serine/threonine protein kinase involved
in the MAPK signalling pathway that regulates cell division, growth,
differentiation and survival. BRAF mutations, such as BRAF V600E, activate
the MAPK signalling pathway which enhances proliferation and cancer cell
survival. BRAF V600E mutated colorectal cancer represents a subset of
colorectal cancer characterized by poor overall survival and limited response
to chemotherapy. Despite multiple studies showing evidence of response to
combined BRAF/EGFR, BRAF/MEK and BRAF/EGFR/MEK inhibition, overall
response rates and median progression-free survival are modest, which
further underlines the necessity for more effective therapies for this specific
patient population.
The major goal of this graduate thesis was to extend the existing
knowledge of signalling pathways and cellular processes regulated by the
BRAF V600E mutation in colon cancer cells. For this purpose, global
proteomic profiling of BRAF V600E mutant (RKO, HT29) versus wild-type
BRAF (HTC116, SW620) colon cancer cells was carried out in the pH ranges
3-10 NL and 4-7 by means of two-dimensional gel electrophoresis followed
by MALDI-TOF/TOF mass spectrometric analysis. In this way, we were able
to identify twenty proteins with statistically significant (p <0.05) changes
in expression levels in BRAF V600E mutated in comparison to wild-type
BRAF colon cancer cells. The majority of identified proteins were involved
in cellular processes important for cancer progression including cellular
communication, cell cycle and growth.
Importantly, this study revealed several novel protein biomarkers
potentially associated with aggressive phenotype of BRAF mutated
colorectal cancer. These include proteins with potential oncogenic role such
as MORN repeat-containing protein 5, zinc finger protein 230, eukaryotic
elongation factor 2 kinase and serine/threonine-protein kinase 40, as well
as those that may act as tumour suppressors including Hepatitis A virus
cellular receptor 2 and Ras-related protein Rab-40A. Further
immunohistochemical analyses are required to ascertain the role of these
proteins in the pathogenesis of BRAF V600E mutated CRC and to assess
their prognostic relevance. In addition, targeting eukaryotic elongation
factor 2 kinase and serine/threonine-protein kinase 40 may prove as a novel
strategy to control behaviour of BRAF mutated CRC cells and to increase
their sensitivity to chemotherapy.
Keywords
MAPK signalni put
BRAF V600E mutirani kolorektalni karcinom
2-DE
MALDI-TOF/TOF MS
Keywords (english)
BRAF kinase
MAPK signalling pathway
BRAF V600E mutated colorectal cancer
2-DE
MALDI-TOF/TOF MS
Language croatian URN:NBN urn:nbn:hr:193:230516 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2018-12-20 08:44:04
