Estudio in vitro e in vivo del rol pro-metastásico de Caveolina-1 en vesículas extracelulares de líneas de cáncer de mama metastásico
Breast cancer is the leading cause of cancer-related deaths in women. Although the incidence of this disease has decreased thanks to the implementation of screening mammograms and application of adjuvant therapies, this decrease does not seem to be enough, since the development of metastasis is stil...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2019 |
| País: | Chile |
| OAI Identifier: | oai:repositorio.anid.cl:10533/236369 |
| Acceso en línea: | https://hdl.handle.net/10533/236369 |
| Access Level: | acceso abierto |
| Palabra clave: | Medicina y Ciencias de la Salud Medicina Básica Otros Temas de Medicina Básica |
| Sumario: | Breast cancer is the leading cause of cancer-related deaths in women. Although the incidence of this disease has decreased thanks to the implementation of screening mammograms and application of adjuvant therapies, this decrease does not seem to be enough, since the development of metastasis is still responsible for more than 90% of deaths associated with breast cancer, among other types of cancer. The progression of tumor cells towards a metastatic state implies the acquisition of characteristics, such as resistance to apoptosis, migration and high invasiveness, etc. Taking into account the latter, it has been described that many of these characteristics are enhanced by the expression of Caveolin-1 (CAV1), thereby implicating this membrane protein in the progression of cancer. Specifically in advanced breast cancer it has been observed that a high expression of CAV1 is associated with a shorter survival of the patient. On the other hand, in vitro studies conducted in our laboratory using the human metastatic breast cancer cell line, MDA-MB-231, have indicated that the silencing of CAV1 leads to a decrease in migration, polarization and focal adhesion turnover in comparison with control MDA-MB-231 cells. The question that arises is how CAV1 may promote migration, invasion and metastasis, considering that this process is very inefficient because less than 0.1% of the disseminated cells successfully establish a metastatic nodule. One possibility may be that CAV1 is present together with other molecules in extracellular vesicles (EVs), which serve as vectors to transport these components to adjacent cells within the same tumor microenvironment and/or to distant sites where they may condition the pre-metastatic niche. Here, it should be noted that EVs from MDA-MB-231 cells reportedly promote migration of non-metastatic MCF-7 human breast cancer cells, but the precise content of these EVs and their role in this process were not defined. This led us to propose the following hypothesis: CAV1 in extracellular vesicles increases the migratory and invasive potential of breast cancer cells in vitro and in a breast cancer xenotransplant model in vivo. Therefore, the main goal of this thesis was to evaluate the migratory and invasive capacity in vitro and metastatic breast cancer cells in vivo in a xenotransplant model exposed to EVs containing CAV1. To address the working hypothesis, the following specific objectives were proposed: (1) To purify and characterize extracellular vesicles from human metastatic and non-metastatic breast cancer cell lines; (2) To study the effect of CAV1-containing EVs on migration and invasion in vitro of human breast cancer cell lines; (3) To evaluate the protein content by mass spectrometry of EVs from the three breast cancer sub lines MDA-MB-231 WT, shC and shCAV1; and finally (4) To evaluate the metastatic potential in vivo in a murine xenotransplant model inoculated intraperitoneally with EVs containing or not CAV1. The results showed that we were able to isolate an EV preparation enriched in vesicles of <200 nm in diameter, which is characteristic of exosomes. The EVs were purified from conditioned media of the human metastatic breast cancer cell lines, MDA-MB-231 and MDA-MB-231(shC), both expressing elevated endogenous levels of CAV1, as well as from cells lacking CAV1, such as MDA-MB-231(shCAV1) cells. This characterization was complemented by transmission electron microscopy of the isolated vesicles, which revealed that vesicles were around 100 nm in diameter. Finally, by western blotting, CAV1 was detected together with exosome markers in vesicles from MDA-MB-231 WT and shC but not in MDA-MB-231(shCAV1) EVs. To evaluate the biological effects of vesicles with or without CAV1 on metastatic or non-metastatic breast cancer cells, migration and invasion parameters of these cells were evaluated following exposure to EVs. Regardless of the cell type that was used as a recipient cell, those cells that were treated with EVs containing CAV1 increased their migratory and invasive potential in comparison with cells that were either not treated or treated with EVs lacking CAV1. Analysis by mass spectrometry revealed the presence of specific proteins related to cell adhesion, such as Cyr61, tenascin (TNC) and S100A9 only in MDA-MB-231 WT and shC EVs but not in EVs from MDA-MB-231 lacking of CAV1. These results were confirmed by western blotting analysis. In order to evaluate the role of CAV1 in EVs in a murine carcinoma model, these vesicles were injected intraperitoneally together with metastatic or non-metastatic breast cancer cells into recipient mice. For animals inoculated with cells plus EVs containing CAV1, the number of tumor cells found in the ascitic fluid generated within the peritoneal cavity was dramatically increased. Also, a substantial increase in the tumor mass in spleen/pancreas and mesentery was observed in these mice compared to those animals that were either not treated, treated only with cells or treated with cells plus EVs that did not contain CAV1. It should be noted that again these effects of CAV1-containing EVs were observed regardless of whether the reciepient breast cancer cell type employed in these experiments expressed CAV1 or not. Thus, the biological effects of these vesicles in the recipient cell appear not to be attributable to CAV1 per se, but rather to depend on differences in the molecular cargos that are incorporated into EVs in the presence of CAV1. |
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