Vrednotenje signalnih poti, ki vključujejo katepsina B in X, v tumorskih matičnih celicah raka dojke

Frlic, Tjaša (author), Kos, Janko (mentor), Mitrović, Ana (co-mentor)

Rak dojke je drugi najpogostejši vzrok smrti zaradi raka pri ženskah na svetu. Smrtnost zaradi raka dojke se je zaradi napredka pri diagnosticiranju in zdravljenju bolezni v zadnjih desetletjih značilno zmanjšala. Kljub temu pri številnih bolnicah ostajata problem neodzivnost na zdravljenje in ponovni pojav bolezni, za kar je odgovorna majhna populacija rakavih celic – tumorske matične celice (TMC). Zato potrebujemo nove terapevtske pristope, ki bi bili učinkoviti tudi proti tej populaciji celic. Poleg obstoječih protitumornih terapij, ki ciljajo diferencirane tumorske celice, bi s ciljanjem TMC lahko dosegli daljše obdobje brez bolezni ali popolno okrevanje bolnic z rakom dojke. Kot obetavni terapevtski tarči sta se izkazali cisteinski lizosomski peptidazi, katepsina B in X, ki sta udeležena v ključnih procesih nastanka in napredovanja raka. Za njihovo uspešno uvedbo v nove terapevtske pristope zdravljenja raka je ključnega pomena tudi razumevanje njihovih molekularnih mehanizmov in učinkov na ključne signalne poti v TMC. TMC smo izolirali iz celičnih linij MDA­-MB-­231, MCF-­10A neoT in MCF7 na podlagi njihove sposobnosti tvorbe tumorskih sfer. Najprej smo po detekciji s specifičnimi protitelesi na membrani po prenosu western pokazali, da je izražanje katepsinov B in X v TMC, izoliranih s tvorbo tumorskih sfer, povečano v primerjavi z diferenciranimi tumorskimi celicami pred izolacijo TMC. Z zaviranjem katepsinov B in X smo nato ovrednotili vpliv na celično signalizacijo v TMC. Pri tem smo določili izražanje izbranih proteinov, ki so udeleženi v ključih signalnih poteh TMC; pGSK3β, ciklin D1, β-­katenin, TGF­β in NF-­κB. Pokazali smo, da z zaviranjem katepsinov B in X pomembno vplivamo na signalni poti Wnt in TGF­β, medtem ko zaviranje katepsinov B in X nima večjega vpliva na signalno pot NF-κB. Nazadnje smo z zaviranjem signalnih kinaz določili vlogo katepsinov B in X v signalnih poteh TMC raka dojke. S prenosom western in določanjem aktivnosti z encimsko kinetiko smo pokazali, da imata katepsina B in X pomembno vlogo v signalnih poteh JAK/STAT, Wnt, MAPK in PI3K ter sta vključena v signalizacijo preko Trk in integrinskih receptorjev. Pokazali smo, da sta katepsina B in X udeležena v številnih signalnih poteh TMC raka dojke. S tem smo prispevali k razumevanju učinka zaviranja katepsinov B in X na ključne signalne poti v TMC, kar je pomembno za razumevanje njihovih molekularnih mehanizmov in možnost uporabe pri protitumorni terapiji.

• Language: Slovenian
• Keywords: rak, tumorske matične celice, signalne poti, katepsin B, katepsin X
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-149361
• Publication date in RUL: 07.09.2023

Secondary language

• Language: English
• Title: Evaluation of signalling pathways which include cathepsins B and X in breast cancer stem cells

Abstract

Breast cancer is the second leading cause of cancer death in women worldwide. Breast cancer mortality has significantly decreased in recent decades as a result of advances in the diagnosis and treatment of the disease. However, in many patients the problem remains resistance to treatment and recurrence of the disease, which may be atributed to a small subset of cancer cells – cancer stem cells (CSCs). In addition to existing anti­tumour therapies which target differentiated tumour cells, targeting CSC could lead to a longer period without disease or a full recovery for breast cancer patients. Lysosomal cysteine peptidases, cathepsins B and X that have important role in different processes of development and progression of cancer, could serve as promising molecular targets. Identification of their targets and effects on key signaling pathways in CSCs is therefore necessary. We isloated CSCs from breast cancer cell lines MDA­-MB-­231, MCF-­10A neoT and MCF7, based on their ability to form tumorspheres. Using western blot analysis, we showed that the expression of cathepsins B and X in breast CSCs, isolated by tumorsphere formation, is increased compared to differentiated cells prior to CSCs isolation. Next, we demonstratedthe effect on cell signaling in breast CSCs by inhibiting cathepsins B and X. For this purpose, we determined the expression of selected proteins, involved in key signaling pathways of breast CSCs; pGSK3β, cyclin D1, β­catenin, TGF­β and NF-­κB. Inhibition of cathepsins B and X had a significant effect on Wnt and TGF-­β signaling pathways, while the effect on the NF­κB signal pathway has not been demonstrated. Additionally, we determined the effect of inhibiton of signal kinases on cathepsins B and X in the signaling pathways of breast CSCs. Using western blot analysis and by determining enzyme activity using enzyme kinetics we demonstrated that cathepsins B and X play an important role in signaling pathways JAK/STAT, Wnt, MAPK and PI3K and are involved also in Trk signaling and integrin receptor signaling. We showed that cathepsins B and X are involved in many signaling pathways of breast CSCs. With that, we contributed to better understanding of the effect of cathepsins B and X inhibition on key signaling pathways in CSCs, which is important for understanding their molecular mechanisms and possibility of use in anti­tumour therapies.

• Keywords: cancer, cancer stem cells, signaling pathway, cathepsin B, cathepsin X