الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 87 |  |  |
| | | from | 87 | | |
Abstract
Triple-negative breast cancer (TNBC) is a subtype of breast cancer (BC) characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), and Her2 protein. Consequently, the proliferation of TNBC is not driven by estrogen, progesterone hormones, or the Her2 protein. As a result, TNBC does not typically respond to hormonal therapy or drugs targeting Her2 protein receptors. TNBC represents approximately 10-20% of breast cancers, displaying a more aggressive nature and a less favorable prognosis compared to other breast cancer types. Studies indicate that TNBC is prone to early metastasis at presentation and recurrence after treatment. It often presents with a higher grade and typically exhibits a basal-like cellular morphology.
Autophagy serves as the primary mechanism facilitating the transportation of diverse cellular cargoes to lysosomes for degradation and recycling. Nonetheless, in cancer, contradictory functions of autophagy emerge, playing roles both in preventing early tumor development and in sustaining the metabolic adaptation of established and metastasizing tumors. Nevertheless, in BC, autophagy may play an important role in tumor progression, although the mechanism by which autophagy regulates metastatic potential still unclear. Autophagy type II programmed cell death has a dual role in cancer cells and is regulated by a network of intricate signaling pathways for instance signal transducer and activator of transcription 3 (STAT3) signaling pathway. This crosstalk between autophagy and other signaling pathways may decide the survival or death of a cell. Evidently, it has been demonstrated the pivotal role of STAT3 which has been widely recognized as a regulator of genes expression which either related to tumorigenic behaviour of breast cancer specially, TNBC; initiation, cell proliferation, progression and metastasis, or regulating the genes expression of several types of signaling pathways including autophagy. Furthermore, STAT3 not only has the capacity to induce the expression of genes associated with cancer but also engages in physical interactions and functional collaboration with other oncogenic transcription factors, contributing to the aggressiveness of triple-negative breast cancer (TNBC).
On the hand, 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 objective of the present study is to evaluate the proposed role of exosomes in the aggressiveness behaviour of TNBC cells. Also, the present work was directed to assess the role of exosomesin modulatingthemolecular mechanisms involved in exhibiting the aggressive behaviour e.g., autophagic machinery through STAT3 signaling pathway in TNBC cells.
To approach the objectives, theblood samples were collected from TNBC patients and exosomes were extracted and characterized the size and shape via scanning electron microscope (SEM) and detecting of protein biomarkers via western blotting. In addition, two types of breast cancer cell lines were utilized; TNBC, MDA-MB231 (masenchymal stem-like) and HCC1806 (basal-like), and NTNBC, MCF7 (Estrogen-receptor-positive). Accordingly, each cell line was divided into the following groups:
Summary and Conclusion
65
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 20μl of EXO.
5. InSTgp:
Cells were treated with IC50 of AG490, STAT3 pathway inhibitor.
6. InST-EXO gp:
InST cells were treated cells with 20μg of EXO.
7. InST-DOXO gp:
InST cells were treated with IC50 of DOXO.
8. InST-EXO-DOXO gp:
InST cells were treated with IC50 of EXO and DOXO.
After treatments, the cells were harvested for downstream applications. , western blotting technique was usedtoevaluate the STAT3 inhibition in each cell line before and AG490 treatment. Additionally, the autophagic machinery was monitored not only by evaluating the overall flux using LC3B protein levels in cells via ELISA but also evaluating the genes expression for BECLIN1, ATG16L1, ATG5 and RAB24 which represent all stages of autophagy was assessed via RT-PCR.
The results of SEM 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 by W.B technique revealed the presence ofCD63, PCD6IP (Alix) and TSG101, indicating the presence of exosomal surface-marker proteins, where CD63 is the most abundant relative to TSG101 and PDCDIP. Meanwhile, the uptake of PKH67 labeled exosomes by MDA-MB231, HCC1806 and MCF7 cells was detected under fluorescent microscope.The FM images showed that the exosomal uptake efficiency was higher in TNBC cells (slightly higher in MDA-MB231 cells than HCC1806 cells) than in NTNBC cells; MCF7. 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 EXO 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 tumorigenesis behaviours.Morphological changes associated with DOXO treatment markedly affected NTNBC cells than TNBC cells. The observed alterations are in consistent with the changes in tumorigenic behavior of BC cells as reflected by proliferation, migration and colonizationespecially in MDA-MB231 cells. 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 InST-TNBC and InST-NTNBC cells revealed different responses compared to EXO treated cells. In InST-MDA-MB231, EXO treatment showed a decrease in the frequency of spindle shaped cells, while in InST-MCF7 cells, the cell aggregations and clumps were disappeared. In contrast, addition of EXO to InST-HCC1806 cells revealed the presence of cells mixture among them small circular shaped cells resemble
Summary and Conclusion
66
hematopoietic stem cell like with increase in the cell viability as well as dead cells. The proliferation and migration of InST-TNBC and InST-NTNBC treated with EXO were less pronounced compared to EXO treated cells. Regarding colonization, InST-TNBC treated with EXO showed a reduction in the colony formation ability while, in InST-NTNBC cells treated with EXO, the ability for colony formation was prohibited. Thus, this may point out to the enhanced anti-tumorigenic effects of AG490 through STAT3 inhibition in both subtypes of BC. Meanwhile, it may provide more evidences the inhibition of STAT3 pathway enhanced the anti-tumor effects of DOXO treatment in BC cells. Moreover, treatment of InST-TNBC cells with EXO-DOXO caused a decrease in the proliferation, migration and colonization of cells compared to that in cells treated with EXO-DOXO only. While, in InST-NTNBC cells, the proliferation and migration were reduced whereas, colonization was completely diminished. These results may point out to the anti-tumorigenic effect of AG490.
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 EXO in strengthen tumorigenic behavior of NTNBC or TNBC.Cell cycle analysis ofDOXO 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 EXO in enhancing the tumorigenic behavior of recipient BC cells especially in induction of chemoresistance of BC cells to DOXO treatment. However, EXO treatment of both InST-TNBC and InST-NTNBC cells revealed an increase in apoptotic cells accompanied by a decrease in proliferative cells compared to EXO treated cells. On the contrary, cell cycle analysis of InST-TNBC cells showed an increase in cell population arrested at G2/M phase associated with the decrease in G0/G1 compared to EXO treated cells. These observations may point out to the enhanced tumorigenic role of EXO in TNBC cells than NTNBC owing either to the origin of EXO or to the type of recipient cells. Meanwhile, treatment of InST-TNBCand InST-NTNBC cells with DOXO showed the prevalence of apoptotic cells which may suggest that inhibition of STAT3 signaling pathway increased breast cancer cell response to the pro-apoptotic effect of DOXO.In addition, apoptotic assay of InST-TNBC and InST-NTNBC cells with EXO-DOXO showed that the majority of cells were in apoptotic phase compared to EXO-DOXO treated cells without inhibition which may point out to the anti-cancer effect of AG490.
The biochemical results showed that MDA-MB231 cells showed up regulation of LC3B, ATG5 and RAB24 which indicate ideal exosomal stimulation to autophagic flux which may take part in increasing the aggressiveness of tumorigenic behavior.Interestingly, HCC1806 cells showed also increase in LC3B level despite down regulation of BECLIN1, ATG16L and RAB24. These results may suggest accumulation of autophagosome which may indicate either increase in autophagyor a blocking of autophagy at the fusion step with lysosomes. Since, it has been reported in previous studies thatBECLIN1 expression regulates coincidentally autophagic process where BECLIN1 downregulation due to the treatment with EXO does not decay the role of EXO in autophagic tumorigenesis. Also, ATG16L overexpression may cause an indirect effect on autophagy where it was noticed that its overexpression inhibits autophagosome. Moreover, RAB24 downregulation which could be
Summary and Conclusion
67
due to slowdown or inhibition of autophagic flux sinceautophagy could potentially be suppressed at any stage of autophagic flux.In MCF7 cells, LC3B level was significant increase with downregulation of ATG16L that may indicate increase in autophagic flux and increase aggressiveness of cells.
On the other hand, DOXO as anticancer treatment in MDA MB231 lead to increase LC3B levels and up regulation of ATG5 expression reflecting the cytoprotective role of autophagy through its stimulation in response to DOXOtreatment. While treatment of HCC1806 cells showed significant downregulation of BECLIN1 and RAB24 only, resulting in slowing down or inhibition of autophagic machinery. Additionally, MCF7 cells showed upregulation in LC3B level and BECLIN1 expression indicating stimulation of autophagic flux.
In EXO-DOXO treated cells, it was observed increase level of LC3B indicating activation of autophagic flux only in MDA-MB231 which reflects the crucial role of EXO regardless of the presence of DOXO which can’t be ignored. EXO-DOXO treatment of HCC1806 and MCF7 cells showed a downregulation in BECLIN1 expression only indicating the slowing down or total suppression of autophagic machinery, as it was supported in previous studies that BECLIN1 deficiency resulted in loss of autophagic vacuole formation. STAT3 pathway either negatively or positively regulates autophagy depending on the differences in cells and/or STAT3 phosphorylation sites. In TNBC cells, InST-EXO cells showed upregulation in the ATG16L expression associated with down-regulation of BECLIN1 and ATG5 expression. The only observed difference was the significant decrease in LC3B level in InST-HCC180 cells reflecting the slowing down or total inhibition of autophagy. These results could be attributed to the aggressive tumorigenic behavior of TNBCs potentiated by EXO treatment which may be autophagy dependent. On contrary in NTNBC cells, InST- EXO cells showed upregulation of all measured autophagic genes expressions indicating the influence of EXO treatment on tumorigenic behavior in NTNBC is autophagy independent effect. However, InST-MDA MB231treatment with DOXO showed downregulation of LC3B level and BECLIN1 expression and upregulation of ATG16L1 and RAB24 expressions which may reflect the slowdown or inhibition of autophagic flux. Furthermore, treatment of InST-HCC1806 cells with DOXO revealed no significant changes in studied genes expressions or LC3B level. Thus, treatment of InST-HCC1806 cells with DOXO has no influence on the autophagic flux. On contrary, InST-MCF7 cells treated with DOXO showed upregulation of all studied genes of autophagic machinery, revealing induction of autophagic machinery.
At least not last comparing to EXO-DOXO treated groups, InST-MDA MB231 treated with EXO-DOXO showed decrease of LC3B level with downregulation of BECLIN1, ATG5 and ATG16L expressions, while InST-HCC1806 showed also decrease in LC3 level with downregulation of ATG5 and RAB24 expressions which may reflect slowdown or suppression of autophagic machinery. On the other hand, EXO-DOXO treatment InST MCF7 cells, showed significant upregulation of all assessed genes associated with significant LC3B level increase that may be induced activation of autophagic machinery. Finally on comparing to InST-DOXO cells, InST-TNBC treated cells with EXO-DOXO showed significant downregulation in BECLIN1 and ATG5 expression without any significant change in LC3B level. While in InST-NTNBC treated cells with EXO-DOXO, didn’t show any significant change in assessed autophagic genes but only showed significant increase in LC3B level. These results may favor the suggestion that the EXO potentiate the aggressive behavior of TNBC via autophagy but not in NTNBC.
Summary and Conclusion
68
In conclusion, the present study showed that the role of TNBC-derived-exosomes may play a part in the aggressiveness of tumorigenic behavior including progression, migration, invasion, induction of drug resistance and metastasis through manipulating several signaling pathways as autophagic machinery and STAT3, implicated in cell cycle progression and tumorigenesis. In this aspect, the findings could be argued that the aggressive behaviour of TNBC potentiated by EXO treatment could be autophagy-dependent while in NTNBC is autophagy independent.