Dual Knockdown of Musashi RNA-Binding Proteins MSI-1 and MSI-2 Attenuates Putative Cancer Stem Cell Characteristics and Therapy Resistance in Ovarian Cancer Cells.

Maria T Löblein,Isabel Falke,Hans Theodor Eich,Fabian M Troschel,Martin Götte,Burkhard Greve

doi:10.3390/ijms222111502

Maria T Löblein, Isabel Falke + Show 4 more

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https://doi.org/10.3390/ijms222111502

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Abstract

In ovarian cancer, therapy resistance mechanisms complicate cancer cell eradication. Targeting Musashi RNA-binding proteins (MSI) may increase therapeutic efficacy. Database analyses were performed to identify gene expression associations between MSI proteins and key therapy resistance and cancer stem cell (CSC) genes. Then, ovarian cancer cells were subjected to siRNA-based dual knockdown of MSI-1 and MSI-2. CSC and cell cycle gene expression was investigated using quantitative polymerase chain reaction (qPCR), western blots, and flow cytometry. Metabolic activity and chemoresistance were assessed by MTT assay. Clonogenic assays were used to quantify cell survival post-irradiation. Database analyses demonstrated positive associations between MSI proteins and putative CSC markers NOTCH, MYC, and ALDH4A1 and negative associations with NOTCH inhibitor NUMB. MSI-2 expression was negatively associated with the apoptosis regulator p21. MSI-1 and MSI-2 were positively correlated, informing subsequent dual knockdown experiments. After MSI silencing, CSC genes were downregulated, while cell cycle progression was reduced. Metabolic activity was decreased in some cancer cells. Both chemo- and radioresistance were reduced after dual knockdown, suggesting therapeutic potential. Dual knockdown of MSI proteins is a promising venue to impede tumor growth and sensitize ovarian cancer cells to irradiation and chemotherapy.

Highlights

Proteins of the Musashi family can be found in normal and tumor cells
We aimed to establish the relationship between the Musashi family and putative cancer stem cells to understand the therapeutic potential of a dual Musashi knockdown in ovarian cancer
Further significant correlations were identified for both Musashi RNA-binding proteins (MSI)-1 and MSI-2 with cancer stem cell-associated genes of the notch signaling pathway: both were negatively correlated with NUMB (Figure 1(B1,B2)) and positively correlated with NOTCH-3 (Figure 1(C1,C2))

Summary

Introduction

Proteins of the Musashi family can be found in normal and tumor cells. As stem cell markers, they regulate numerous targets by binding messenger ribonucleic acids (mRNAs) via specific RNA-binding motifs. Musashi consists of two variants, Musashi-1 (MSI-1) and Musashi-2 (MSI-2) Both proteins have been shown to be about 90% homologous and are closely related in their RNA-binding specificity [2,3]. Dysregulation of either or both of these highly conserved proteins can lead to cellular dysfunction, including instability and tumorigenesis in a wide array of different cancer entities [6,7] Expression of both Musashi proteins has been shown to be negatively associated with overall survival in cancer patients [8,9]. Recent data of our group suggest a radioprotective role of Musashi in triple negative breast cancer as siRNA-based knockdown radiosensitized cells [10] These effects were mainly caused by modulation of notch signaling pathway [11], cell cycle signaling [12], and DNA damage repair [13]

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