الفهرس 
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Abstract
Triple Negative Breast Cancer (TNBC) is a subgroup of breast cancer (BC) that accounts for approximately 15-20 % among BC. TNBC have high heterogeneity and further can be classified into 6 subtypes and characterized by its aggressive behavior that is associated with large tumor size, lymph node involvement, high histological grade, poor prognosis, low survival rate and lack of targeted therapy.
The dysregulation/dysfunction of signaling pathways that modulates the normal development and function of the mammary gland have a significant role in cancer progression as they are involved in cancer cell proliferation, differentiation and survival. Moreover, FOXO pathway is “double-edged swords” dualistically involved in the regulation of various steps of carcinogenesis and metastasis. As well, Wnt/β-catenin signaling pathway is a central signaling pathway which exerting crucial roles in tumorigenesis behaviour of cancer, including breast cancer, and their response to therapy. It is found a cross talk between the WNT and FOXO signaling pathways in cancer development and chemoresistance. On the other hand, Silent information regulator (SIRT1) and toll like receptor 4 (TLR4) were found to co-operate with FOXO proteins and to play a critical role in metastasis process and the treatment of BC.
On the other hand, tumor microenvironment may be stimulating drug resistance by preventing drugs accumulation in cancer cells for instance exosomes. Exosomes are nano-sized vesicles that carry cargoes of bioactive molecules, such as nucleic acids, proteins, lipids, and metabolites. They are involved in cell-cell communication by transferring molecular cargoes to recipient cells. Accordingly, the tumorigenic role of exosomes in enhancing the aggressive of TNBC has been suggested.
Thus, the main objective of the present study is to evaluate the role of exosomes in potentiating the tumorigenic behavior of TNBC cells. The proposed role of exosomes was evaluated in the view of the influences of down-regulation of FOXO3 pathway in TNBC on WNT signaling pathway and cell death mechanism. To approach the objective of the present study two types of breast cancer cell lines were utilized; TNBC cells, MDA-MB231 (mesenchymal stem-like) and HCC1806 (basal-like), and NTNBC cells, MCF7 (Estrogen-receptor-positive). Accordingly, each cell line was divided into the following groups:
1. Un-Treated gp: Untreated control cells
2. EXO gp: Cells were treated with 20µg/protein of exosomes (EXO); depending on the exosomes up-take test.
3. DOXO gp: Cells were treated with IC50 of doxorubicin (DOXO)
4. EXO-DOXO gp: Cells were treated with IC50 of DOXO and 20µg of EXO.
5. InFX gp: Cells were treated with IC50 of Sirtinol, FOXO pathway inhibitor.
6. InFX-EXO gp: InFX cells were treated cells with 20µg of EXO.
7. InFX-DOXO gp: InFX cells were treated with IC50 of DOXO.
8. InFX-EXO-DOXO gp: InFX cells were treated with IC50 of DOXO and 20µg of EXO.
In the present study, blood samples were collected from TNBC patients and exosomes were extracted characterized by scanning electron microscope (SEM) and detecting of exosomal protein biomarkers by western blotting. SEM analysis of exosomes revealed the presence of exosomes with their characteristic biconcave shape and different sizes (30-300 nm), as well as the large size subclass was predominant. Meanwhile, characterization of exosomal protein biomarkers revealed the presence ofCD63, PCD6IP (Alix) and TSG101, where CD63 is the most abundant relative to TSG101 and PDCDIP proteins. The uptake of PKH67 labeled exosomes by MDA-MB231, HCC1806 and MCF7 cells was detected under fluorescent microscope (FM).The FM images showed that the exosomal uptake efficiency was higher in TNBC cell lines than in NTNBC cell line. The exosomal uptake was depended on the origin and recipient cells.
Morphologically, the treatment of TNBC cells with EXO lead to the elongation of the spindle shape structure of epithelial cell However, the treatment of NTNBC with exosomes resulted in cellular aggregates. Moreover, EXO treatment of both to TNBC and NTNBC cells resulted in an increase in cell proliferation, migration and colonization cells. All of which may suggest the ability of TNBC derived exosomes of significantly increasing recipient cells’ invasion potential via transferring functional cargo molecules to those cells promoting cell proliferation, colonization and migration. Morphological changes associated with DOXO treatment markedly affected NTNBC cells; MCF7 than TNBC cells, MDA-MB231 and HCC1806, respectively. The observed alterations are in consistent with the changes in tumorigenic behavior of BC cells as reflected by proliferation, migration and colonization especially in MDA-MB231.However, EXO-DOXO treatment TNBC cells with still showed elongated spindle shape structures compared to DOXO treated cells. In the same context, treatment of TNBC and NTNBC cells with EXO-DOXO increased cell proliferation, migration and colonization of BC cells. All of these may add more evidence and support for the implication of exosomes in chemoresistance of BC cells to DOXO treatment.
Interestingly, EXO treatment of InFX-TNBC and InFX-NTNBC cells revealed same responses compared to EXO treated cells where no morphological alterations were observed. The proliferation, migration and colonization of InFX-BC cells treated with EXO were pronounced reduced compared to EXO treated cells. These observations supported the suggestion that EXO may enhance the tumorigenic behaviour of TNBC or NTNBC cells via FOXO signaling pathway. Moreover, InFX-TNBC and InFX-NTNBC cells, treatment with DOXO morphologically reduced the number of viability compared to DOXO treated cells. As well, the effects of DOXO treatment on the proliferation, migration and colonization in InFX-TNBC and InFX-NTNBC cells were observed. Thus, these results may provide more evidence for the anti-tumor effects of the combination sirtinol and DOXO in BC cells especially for TNBC. Regarding treatment of InFX-BC with EXO-DOXO and compared to EXO-DOXO treated cells, the morphological alterations and the reduction of proliferation, migration and colonization were observed and could be attributed to the combined anti-tumor effects of sirtinol and DOXO. Whereas, the effects of EXO-DOXO treatment compared to that of DOXO treatment in the Morphological alterations and the observed changes in the tumorigenic behaviour could be attributed to the influences of the exosome.
Regarding cell cycle analysis of TNBC and NTNBC cells, treated with EXO revealed an increase in cell population arrested at the S-phase. Meanwhile, the apoptotic assay revealed an increase in necrotic cells. These observations may suggest tumor progression and aggressiveness thus throwing more lights on the roles of exosomes in strengthen tumorigenic behavior of NTNBC or TNBC. Cell cycle analysis of DOXO treated TNBC cells revealed an increase in cell population arrested at Go/G1 denoting its pro-apoptotic effect. Also, apoptosis assay showed an increase in the percentage of late apoptotic and necrotic cells in MDA-MB231, whereas in HCC1806 cells only necrosis was observed. However, treatment of NTNBC cells with DOXO led to the increase in cells arrested at Sub G1 revealing its pro-apoptotic effect. The results of cell cycle analysis of TNBC cells treated with EXO-DOXO may suggest cell cycle progression. Moreover, the results and observations may support more the role of exosomes in enhancing the tumorigenic behavior of recipient BC cells especially in induction of chemoresistance of BC cells to DOXO treatment.
Analysis of cell cycle in InFx-TNBC cells treated with exosomes showed a decrease in cell population arrested at the G0/G1 phase and an increase in G2/M phase compare EXO-treated cells. Also, apoptosis assay analysis showed a decrease in proliferated cells and an increase in apoptotic cells. These confusing results may support more the suggestion that exosomes play an important role in enhancing the aggressive tumorigenic behaviour of TNBC possibly through FOXO3 signaling pathway. As for InFX-NTNBC cells, an increase in G0/G1 cells arrest with a decrease in cells arrested in G2/M phase. Also, an increase in apoptotic cells were noticed which may denote the role of exosomes in potentiating the tumorigenic attitude of NTNBC through FOXO3 signaling. For DOXO treatment, cell cycle analysis of InFX-TNBC cells treated with DOXO revealedno dramatic alterations. However, apoptotic assay in InFX-TNBC revealed an increase in apoptotic cells associated with a decrease in proliferated cells. These observations may point out to that inhibition of Sirt1 may potentiate the pro-apoptotic effect of DOXO. Meanwhile, in InFX-MCF7 cells the cell cycle and apoptotic analysis may refer to the pro-apoptotic effect of the combination treatment of DOXO and sirtinol. Interestingly, treating of InFX-TNBC or InFX-NTNBC cells comparing to EXO-DOXO treated cells, with EXO-DOXO showed no alterations changes in cell cycle phases while apoptotic assay revealed an increase in apoptotic cells associated with a decrease in proliferated cells. In InFX-NTNBC showed an increase in early apoptotic cells. These observations explained the predominant effect of DOXO, as anti-tumor, combined with sirtinol to overcome chemoresistance characteristics exerted by exosomes.
The biochemical showed that the genes expressions of WNT signaling pathway markers in MDA-MB231, HCC1806 and MCF7 cells after EXO and/or DOXO also before and after FOXO pathway inhibition may point out to the crucial and important role of exosomes in potentiating the tumorigenic behavior, metastasis and chemoresistance of BC especially in TNBC through signaling pathways. The results revealed that exosomes showed that exosomes may have a significant role in manipulating several signaling pathways implicated in tumorigenesis such as WNT/β-catenin signaling pathways mediated by FOXO.
In conclusion, the present study is an attempt to throw more lights on role of exosomes in enhancement and potentiating of tumorigenic behaviour of TNBC. Also, the study was carried out to consider the mechanism(s) through which exosomes exhibit their tumorigenic role in TNBC. In this aspect, the involvement of Wnt signaling pathway mediated by FOXO pathway was investigated. Thus, the results of the present investigation may provide more evidences and support the crucial and important role of exosomes in exhibiting aggressive behaviour characteristics and drug resistance of BC, generally, and specifically in TNBC.