Breast cancer, a malignant tumor originating from mammary epithelial tissue, is the predominant form of cancer in women and the second-leading cause of female cancer death worldwide. Despite an increased rate of early detection of the disease and improvement in the clinical management of patients, the number of women who die from this disease still remains very high due to tumor dissemination (metastasis) and relapse after initial successful therapy.
Accumulating evidence has suggested that cell-to-cell communication between cancer cells and the tumor microenvironment represents a key driver in tumor progression, but how this crosstalk is regulated is largely unknown. A tumor is surrounded and infiltrated by different cell types (i.e., immune cells, tumor stroma, blood vessels, and epithelial cells), which together contribute to the formation of the tumor microenvironment. The tumor microenvironment is regulated and shaped by the cancer cells and contributes to tumor progression and metastatic cascade. In this scenario, trying to understand how these cells communicate with each other is becoming more and more important, and extracellular vesicles (EVs) have obtained growing attention since they appeared as essential players in the initiation, progression, and metastatic processes in breast cancer.
EVs are naturally released membrane vesicles that can exert broad biological functions and influence the microenvironment via transfer of bioactive molecules and genetic information to neighboring cells, as well as distant sites. EVs have been implicated in several physiological and pathological processes such as inflammation, immune disorders, neurological diseases, and cancer. Although EVs are produced by all cell types under physiological conditions, their production is strongly increased in cancer cells.
Tumor EVs have been implicated in several tumorigenic functions, including the dissemination of cancer cells to distant sites. Accordingly, recent evidence has highlighted how tumor-derived EVs contribute to multiple steps in the metastatic cascade. Hypoxia, a reduction in the normal oxygen level in the tissue, is a hallmark of malignancy and is considered a nearly universal driver of tumor progression and metastasis, including in breast cancer. Recent findings have also demonstrated that tumor cells produce a higher number of EVs under hypoxic conditions and that hypoxic stress affects EV content and function.
Study Details
Our study published in Developmental Cell (2020; doi: 10.1016/j.devcel.2020.07.014) demonstrates that breast cancer cells in low-oxygen conditions (hypoxia) produce EVs that contain messages able to induce oncogenic changes in surrounding normal epithelial cells. Accordingly, normal epithelial cells exposed to hypoxic EVs acquire migratory ability in vitro, which is a characteristic of tumor cells.
We cultured breast cancer cells in hypoxic conditions to mimic the tumor microenvironment and we investigated the role of EVs produced in this condition. To analyze the effects of EVs on surrounding cells, we cocultured them with normal epithelial cells.
We observed that EVs produced by hypoxic breast cancer cells induce a reprogramming of mitochondrial shape and position within the cells, driving cell motility and invasion.
To elucidate the messages vehiculated by EVs, our team analyzed the changes in gene expression in the normal recipient cells after exposure to vesicles produced in hypoxic conditions. We observed an effect on different pathways involved in cell motility, survival, and cell-to-cell contact. These pathways are typically activated in cancer cells, because they are fundamental for tumor initiation and progression. This observation suggests that cancer cells reprogram normal cells to behave as cancer cells.
Interestingly, we also demonstrated that the integrin-linked kinase (ILK) protein is the main cargo in EVs and that all the described changes in normal cells are due to ILK activation. EVs produced by hypoxic breast cancer cells packed ILK and transferred it to the recipient cells that normally don't express it. When internalized in normal recipient cells, ILK activates its downstream targets exerting its function as it would in cancer cells.
Finally, using a 3-D model of normal mammary gland development, we demonstrated that the addiction of EVs produced by hypoxic breast cancer cells disrupts the mammary gland architecture. A single addition of hypoxic EVs deregulates the apical-basal polarity, conferring multiple traits of oncogenic transformation, including decrease in cell death, increase in cell proliferation, and expression of epithelial-to-mesenchymal transition (EMT) markers. EMT is a process that allows poorly migrating epithelial cells to switch to highly migrating mesenchymal cells and has a central role in the early phases of metastatic dissemination.
Our findings suggest that cancer cells can communicate using EVs both locally and with distant sites, supporting tumor progression and the metastatic cascade. Furthermore, our data point to the central role of ILK and mitochondria reprogramming in breast cancer progression. This further suggests that therapeutic targeting of ILK may provide a novel strategy to disrupt early pro-tumorigenic changes in the microenvironment, as well as mechanisms of metastatic competence. Targeting ILK may contribute to complete cancer eradication after surgical treatment and to prevention of relapse.
IRENE BERTOLINI, PHD, is a postdoctoral fellow in the Altieri Laboratory at The Wistar Institute in Philadelphia.