Exosomes as Educators
Exosomes are small secreted vesicles with the ability to mediate cell–cell communication through the horizontal transfer of cellular factors, such as mRNAs, microRNAs, and proteins. Recent studies have highlighted an emerging role for exosomes as key regulators of tumorigenesis and metastasis; however, the underlying mechanisms by which exosomes promote these processes remain poorly understood. In order to elucidate the molecular and cellular function of tumor-derived exosomes, Peinado and colleagues investigated the role of exosomes in metastatic melanoma. The authors isolated exosomes from the plasma of patients with melanoma and found that exosome protein content was significantly higher in patients with metastatic stage IV disease. Using mass spectrometry to analyze exosomal protein content in a panel of metastatic mouse and human melanoma cell lines, the authors identified an exosome signature consisting of tryosinase-related protein-2 (TYRP2), very late antigen 4 (VLA-4), heat-shock protein 70 (HSP70), and heat-shock protein 90 (HSP90) that was enriched in the exosomes of individuals with stage IV disease and showed that one of these factors, TYRP2, had the capacity to predict future metastatic disease in a retrospective analysis of stage III melanoma patients. To test whether exosomes are functionally relevant to disease progression, the authors next injected tumor-derived exosomes into mice and monitored their effects on primary tumor growth and metastasis. Strikingly, exosomes derived from metastatic melanoma cell lines dramatically increased the metastatic burden by 240-fold in the lung and promoted distribution of metastases to additional tissues. The authors show that these intravenously injected exosomes accumulated in common sites of melanoma metastasis, causing changes in the expression of genes involved in extracellular matrix remodeling, such as S100A8 and S100A9, and promoting vascular leakage. In addition, bone marrow transplantation studies revealed that tumor-derived exosomes could promote metastasis through their ability to drive bone marrow–derived progenitor cells toward a provasculogenic c-Kit+ Tie2+ phenotype. To identify the factors responsible for this “exosomal education” of bone marrow–derived cells, the authors compared the proteomic profiles of exosomes from highly metastatic and poorly metastatic cell lines. They showed that one candidate from this screen, the Met proto-oncogene, was present in mouse bone marrow progenitor cells after exosomal treatment and was also elevated in circulating c-Kit+ Tie2+ progenitor cells of patients with stage IV melanoma. Reducing Met levels in exosomes through short hairpin RNA–mediated silencing decreased the levels of vasculogenic precursors in the bone marrow and blood and significantly blocked the prometastatic properties of tumor-derived exosomes. Finally, the authors provide molecular insight into the mechanisms controlling exosome production, showing that the Ras-related RAB proteins, RAB27A, RAB5B, and RAB7, are overexpressed in melanoma cell lines producing high concentrations of exosomes and demonstrating that targeted knockdown of Rab27a is sufficient to reduce exosome secretion and block metastasis. Collectively, these results bring critical insight into a clinically relevant mechanism through which tumor cells promote metastatic colonization through exosomal cross-talk and provide evidence that this process can be exploited as a therapeutic target.
miR34a Regulates PDGFR and TGF-β and Functions as a Tumor Suppressor in Brain Cancer
Transcriptomal profiling of malignant gliomas (high-grade glioblastoma multiforme tumors) identifies 2 principle tumor types, less aggressive proneural tumors and more aggressive mesenchymal tumors. To identify candidate microRNAs (miRNA) that could discriminate between these 2 groups, Genovese and colleagues performed computational network modeling of miRNA and mRNA levels in human gliomas that were available through the Cancer Genome Anatomy Project. They further layered on copy number data and negative correlations between levels of specific miRNAs and mRNAs. To reduce the complexity of these networks, they next identified seed sequences on identified mRNAs that represented targets for specific miRNAs. Eight miRNAs emerged from this analysis that could discriminate between proneural and mesenchymal glioblastoma tumors. Among these miRNAs, miR34a localized to a region of frequent loss in glioblastoma multiforme tumors and was chosen for further studies. The authors showed that tumors in mice engineered to delete both p53 and PTEN in glial cells displayed proneural character and that miR34a functioned as a tumor suppressor in these animals. Mechanistic studies showed that miR34 could regulate both platelet-derived growth factor receptor (PDGFRA) and SMAD3, with the former clearly implicated in gliomagenesis and the latter a key component of TGF-β signaling. In addition, in patients with proneural glioblastoma, low levels of miR34A were correlated with improved survival. These studies correlate genetic and bioinformatics analyses of large human datasets to identify miR34a as a putative tumor suppressor in proneural glioma and suggest restoration of miR34a as a therapeutic strategy.
TP53 Mutation and Chemotherapy Response in Breast Cancer: A Paradoxical Relationship
The relationship of TP53 mutation in breast cancer with response to chemotherapy is unclear. Some studies suggest a poorer response in the setting of TP53 mutation, whereas other studies come to the opposite conclusion. In this report, the authors describe their use of a mouse model of breast cancer to address this question. MMTV-Wnt1 mammary tumors with mutant p53 showed a better clinical response compared with tumors with wild-type p53. In wild-type p53, doxorubicin induced a senescence-like phenotype, allowing tumor regrowth after chemotherapy was discontinued. In contrast, following chemotherapy, p53 mutant tumors did not undergo a growth arrest. However, the mitotic figures that accumulated were abnormal and led to cell death by mitotic catastrophe. The p53 mutant MMTV-Wnt1 tumors also showed a significant increase in both cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive cells after doxorubicin treatment. By comparison, wild-type p53 tumors did not exhibit a similar level of apoptosis following treatment. The presence of wild-type p53 still mediated arrest and inhibited drug response even in the context of p53 heterozygosity or in the absence of the p53 effector gene p21. With respect to the mechanism that accounted for these differences in drug response, the authors conducted a cytokine screen and identified p53-dependent differences in the expression of specific cytokines, including Eotaxin (an eosinophil-selective chemokine) and, to a lesser extent, Cxcl5 and Rantes. They hypothesize that cytokine secretion from senescent wild-type p53 cells could induce proliferation in neighboring nonsenescent cells. Taken together, these results suggest the interesting concept that inactivation of p53 as a therapeutic approach could prevent a senescent-like phenotype in tumor cells and promote mitotic catastrophe and apoptosis, leading to improved chemotherapy response.
Myeloid Cells in Pancreatic Adenocarcinoma
Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy. These cancers have a very dense stromal reaction with many inflammatory leukocytes that are thought to contribute to tumor growth. Bayne and colleagues present evidence that granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates myeloid cells in the tumor microenvironment of PDA. Using a genetic mouse model of PDA, the authors show how malignant cell-derived GM-CSF drives the accumulation of Gr-1+ CD11b+ myeloid cells as part of the cancer-associated inflammatory reaction. The protumor myeloid cells, also known as myeloid-derived tumor suppressor cells (MDSC), are in turn able to inhibit the action of antigen-specific T cells. Abrogation of malignant cell–produced GM-CSF when pancreatic cells are transplanted subcutaneously into mice inhibits the recruitment of Gr-1+ CD11b+ cells to the tumor microenvironment and blocks tumor development, an effect that is dependent on CD8+ T cells. In human PDA, immunohistochemistry staining showed a majority of malignant cells to be positive for GM-CSF. MDSCs were abundant in the mouse PDA model; as many as 20% to 30% of all cells in tumor and spleen were Gr-1+CD11b+, emphasizing the importance of these cells in this malignancy and others. This study highlights the potential for targeting soluble factors such as GM-CSF that regulate the substantial MDSC population alongside other treatments that target the malignant cells or other stromal components. Whether anti–GM-CSF antibodies or other GM-CSF antagonists would be sufficient to achieve a clinical response in the setting of advanced human PDA remains to be seen.
Nonangiogenic Face of VEGF
VEGF/vascular permeability fac-tor (VEGF/VPF, VEGF-A) is a key orchestrator of both physiologic and pathologic angiogenesis, including tumor angiogenesis. VEGF signals through VEGF receptor 1/2 and Neuropilin-1 (NRP-1) receptors and has become a key therapeutic target for many cancers. However, other than extensive studies that have investigated its proangiogenic properties, the role of VEGF in directly regulating tumor growth independent of its proangiogenic activity is not well defined. Cao and colleagues have shown that knockdown of VEGF from several renal cell carcinoma cell lines only expressing NRP-1 receptors significantly reduced tumor growth in vivo. This reduced tumor growth was correlated with increased expression of Ksp-cadherin (a marker of mature kidney epithelial cells)/E-cadherin, downregulation of α-SMA/Snail, and reversal of the differentiation phenotype of the mature kidney cancer cells. Remarkably, the decrease in the in vivo tumor growth was not due to the reduced angiogenesis in the in vivo setting, as shown using a relevant Tet-On-VEGF short hairpin RNA and RFP/GFP expression system. The authors further demonstrated in these cells that both VEGF and NRP-1 in concert activated Ras and downstream extracellular signal-regulated kinase 1/2 and AKT signaling pathways to promote tumor growth. Thus, VEGF can mediate tumor growth through a nonangiogenic switch, resulting in dedifferentiation of tumor cells, thereby expanding its affects on cancer in addition to its established proangiogenic function.
Note: Breaking Advances are written by Cancer Research Editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.
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