Tumor dormancy is a highly prevalent stage in cancer progression. We have previously generated and characterized in vivo experimental models of human tumor dormancy in which micro-tumors remain occult until they spontaneously shift into rapid tumor growth. We showed that the dormant micro-tumors undergo a stable microRNA (miRNA) switch during their transition from dormancy to a fast-growing phenotype and reported the identification of a consensus signature of human tumor dormancy-associated miRNAs (DmiRs). miRNA-190 (miR-190) is among the most upregulated DmiRs in all dormant tumors analyzed. Upregulation of miR-190 led to prolonged tumor dormancy in otherwise fast-growing glioblastomas and osteosarcomas. Here we investigate the transcriptional changes induced by miR-190 expression in cancer cells and show similar patterns of miR-190 mediated transcriptional reprogramming in both glioblastoma and osteosarcoma cells. The data suggests that miR-190 mediated effects rely on an extensive network of molecular changes in tumor cells and that miR-190 affects several transcriptional factors, tumor suppressor genes and interferon response pathways. The molecular mechanisms governing tumor dormancy described in this work may provide promising targets for early prevention of cancer and may lead to novel treatments to convert the malignant tumor phenotype into an asymptomatic dormant state.
Micro-tumors can remain dormant for prolonged periods of time before they switch and enter the rapid growth phase. This initial stage in tumor progression is clearly understudied. In spite of high prevalence, significant clinical implications and increased interest by the research community, tumor dormancy is still poorly understood. The topic of tumor dormancy also suffers from a lack of definition and an agreed upon terminology to describe it. Additionally, the number of reproducible experimental models available for studying indolence of human micro-tumors is quite limited. Here, we describe the development of a general class of in vivo models of indolent human tumors and how these models can be used to elucidate molecular and cellular mechanisms involved in the regulation of dormancy. The models consist of human tumor cell lines that form microscopic cancerous lesions in mice. Although these lesions contain viable and fully malignant cancer cells, the tumors do not expand in size but remain occult for prolonged periods until they eventually spontaneously switch and become fast-growing tumors. Consistent with Judah Folkmans vision that tumors will remain occult and microscopic until they acquire the ability to recruit new and functional blood vessels, the dormancy period of the micro-tumors is associated with impaired angiogenic capacity. Such models can be used for dissecting the host and the tumor-derived regulatory mechanisms of tumor dormancy. Understanding the process by which dormant tumors can overcome growth constraints and emerge from dormancy, resuming size expansion, may provide insights into novel strategies to prolong the dormancy state or to block tumor formation in the early stages, before they are physically detected or become symptomatic.
Evidence suggests that dormant, microscopic tumors are not only common, but are highly prevalent in otherwise healthy individuals. Due to their small size and non-invasive nature, these dormant tumors remain asymptomatic and, in most cases, undetected. With advances in diagnostic imaging and molecular biology, it is now becoming clear that such neoplasms can remain in an asymptomatic, dormant stage for considerable periods of time without expanding in size. Although a number of processes may play a role in thwarting the expansion of microscopic tumors, one critical mechanism behind tumor dormancy is the ability of the tumor population to induce angiogenesis. Although cancer can arise through multiple pathways, it is assumed that essentially most tumors begin as microscopic, non-angiogenic neoplasms which cannot expand in size until vasculature is established. It is now becoming clear that cancer does not progress through a continuous exponential growth and mass expansion. Clinical cancer is usually manifested only in late, unavoidably symptomatic stages of the disease when tumors are sufficiently large to be readily detected. While dormancy in primary tumors is best defined as the time between the carcinogenic transformation event and the onset of inexorable progressive growth, it can also occur as minimal residual or occult disease from treated tumors or as micro-metastases. The existence of dormant tumors has important implications for the early detection and treatment of cancer. Elucidating the regulatory machinery of these processes will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for the development of dormancy-promoting tumor therapies. Despite the high prevalence of microscopic, dormant tumors in humans and the significant clinical implications of their early detection, this area in cancer research has, to date, been under-investigated. In this mini review observations, models and experimental approaches to study tumor dormancy are summarized. Additionally, analogies and distinctions between the concepts of "tumor dormancy" and that of the "cellular dormancy" of tumor cells, as well as between the "exit from tumor dormancy" and the "onset of the angiogenic switch" are discussed.
Dendritic cells (DCs)--immunomodulatory cells that initiate adaptive immune responses--have recently been shown to exert proangiogenic effects when infiltrating the tumor microenvironment. As tumors that escape immune surveillance inhibit DC maturation, we explored whether maturation status determines their ability to promote angiogenesis and whether angiogenesis depends on the presence of DCs. Using mouse xenograft models of human tumors, we show that fast-growing "angiogenic" tumors are infiltrated by a more immature DC population than respective dormant avascular tumors. Accordingly, supplementation of immature DCs, but not mature DCs, enhanced tumor growth. When DCs were mixed with Matrigel and injected subcutaneously into mice, only immature DCs promoted the ingrowth of patent blood vessels. Notably, depletion of DCs in a transgenic mouse model that allows for their conditional ablation completely abrogated basic fibroblast growth factor-induced angiogenesis in Matrigel plugs, and significantly inhibited tumor growth in these mice. Because immature DCs actively promote angiogenesis and tumor growth, whereas DC maturation or ablation suppresses this response, we conclude that angiogenesis is dependent on the presence of immature DCs. Thus, cancer immunotherapies that promote DC maturation may act by both augmenting the host immune response to the tumor and by suppressing tumor angiogenesis.
Clinical trials with antiangiogenic agents have not been able to validate plasma or serum levels of angiogenesis regulators as reliable markers of cancer presence or therapeutic response. We recently reported that platelets contain numerous proteins that regulate angiogenesis. We now show that accumulation of angiogenesis regulators in platelets of animals bearing malignant tumors exceeds significantly their concentration in plasma or serum, as well as their levels in platelets from non-tumor-bearing animals. This process is selective, as platelets do not take up a proportional amount of other plasma proteins (eg, albumin), even though these may be present at higher concentrations. We also find that VEGF-enriched Matrigel pellets implanted subcutaneously into mice or the minute quantities of VEGF secreted by microscopic subcutaneous tumors (0.5-1 mm(3)) result in an elevation of VEGF levels in platelets, without any changes in its plasma levels. The profile of other angiogenesis regulatory proteins (eg, platelet-derived growth factor, basic fibroblast growth factor) sequestered by platelets also reflects the presence of tumors in vivo before they can be macroscopically evident. The ability of platelets to selectively take up angiogenesis regulators in cancer-bearing hosts may have implications for the diagnosis and management of many angiogenesis-related diseases and provide a guide for antiangiogenic therapies.
Tumor dormancy has important implications for early detection and treatment of cancer. Lack of experimental models and limited clinical accessibility constitute major obstacles to the molecular characterization of dormant tumors. We have developed models in which human tumors remain dormant for a prolonged period of time (>120 days) until they switch to rapid growth and become strongly angiogenic. These angiogenic tumors retain their ability to grow fast once injected in new mice. We hypothesized that dormant tumors undergo a stable genetic reprogramming during their switch to the fast-growing phenotype. Genome-wide transcriptional analysis was done to dissect the molecular mechanisms underlying the switch of dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors. A consensus expression signature distinguishing all four dormant versus switched fast-growing tumors was generated. In alignment with our phenotypic observation, the angiogenesis process was the most significantly affected functional gene category. The switch of dormant tumors was associated with down-regulation of angiogenesis inhibitor thrombospondin and decreased sensitivity of angiogenic tumors to angiostatin. The conversion of dormant tumors to exponentially growing tumors was also correlated with regulation and activation of pathways not hitherto linked to tumor dormancy process, such as endothelial cell-specific molecule-1, 5-ecto-nucleotidase, tissue inhibitor of metalloproteinase-3, epidermal growth factor receptor, insulin-like growth factor receptor, and phosphatidylinositol 3-kinase signaling. Further, novel dormancy-specific biomarkers such as H2BK and Eph receptor A5 (EphA5) were discovered. EphA5 plasma levels in mice and mRNA levels in tumor specimens of glioma patients correlated with diseases stage. These data will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for development of dormancy-promoting tumor therapy strategies.
Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant tumors have an impaired angiogenesis potential both in vitro and in vivo and have a slower invasion capacity. This work demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence, and changes in rate of tumor progression.
Tumor dormancy refers to a critical stage in cancer development in which tumor cells remain occult for a prolonged period of time until they eventually progress and become clinically apparent. We previously showed that the switch of dormant tumors to fast-growth is angiogenesis dependent and requires a stable transcriptional reprogramming in tumor cells. Considering microRNAs (miRs) as master regulators of transcriptome, we sought to investigate their role in the control of tumor dormancy. We report here the identification of a consensus set of 19 miRs that govern the phenotypic switch of human dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors to fast-growth. Loss of expression of dormancy-associated miRs (DmiRs, 16/19) was the prevailing regulation pattern correlating with the switch of dormant tumors to fast-growth. The expression pattern of two DmiRs (miR-580 and 190) was confirmed to correlate with disease stage in human glioma specimens. Reconstitution of a single DmiR (miR-580, 588 or 190) led to phenotypic reversal of fast-growing angiogenic tumors towards prolonged tumor dormancy. Of note, 60% of angiogenic glioblastoma and 100% of angiogenic osteosarcoma over-expressing miR190 remained dormant during the entire observation period of ? 120 days. Next, the ability of DmiRs to regulate angiogenesis and dormancy-associated genes was evaluated. Transcriptional reprogramming of tumors via DmiR-580, 588 or 190 over-expression resulted in downregulation of pro-angiogenic factors such as TIMP-3, bFGF and TGFalpha. In addition, a G-CSF independent downregulation of Bv8 was found as a common target of all three DmiRs and correlated with decreased tumor recruitment of bone marrow-derived CD11b+ Gr-1+ myeloid cells. In contrast, antiangiogenic and dormancy promoting pathways such as EphA5 and Angiomotin were upregulated in DmiR over-expressing tumors. This work suggests novel means to reverse the malignant tumor phenotype into an asymptomatic dormant state and may provide promising targets for early detection or prevention of cancer.
For many decades, it has been appreciated that tumor progression is not monotonic, and development of a cancer cell does not equate to inevitable cancer presentation in the clinic. Tumor progression is challenged by numerous intrinsic and extrinsic bottlenecks that can hold the tumor in dormant stages for prolonged periods. Given the complex, multiscale nature of these bottlenecks, the Center of Cancer Systems Biology organized a workshop on critical issues of systems biology of tumor dormancy. The program for the meeting this past July, chaired by N. Almog and H. Enderling, included discussions and interactive breakout sessions on regulation of tumor dormancy by angiogenesis, tumor-immune system interactions, cancer stem cell kinetics, and cell signaling pathways. Three important conclusions emerged from the meeting. The first was the urgent need to differentiate between tumor cell and tumor population dormancy of the primary tumor and metastatic deposits, the second was the continued need for interdisciplinary dialogs, and the third was the need to bring cross-scale mechanistic thinking to the field to achieve a more robust understanding of tumor dormancy and its clinical implications.
Although escape from tumor dormancy has long been recognized as an important problem in the treatment of cancer, the molecular and cellular regulators underlying this transition remain poorly understood. The inability of the cancer cells to induce a complete and successful process of angiogenesis can result in tumor dormancy. In this case, the acquisition of sufficient angiogenic potential will result in the escape from indolence and in the initiation of tumor mass expansion. This stage in disease progression is known as the angiogenic switch. It is now becoming clear that the induction of the angiogenic switch is controlled by dynamic and complex biological processes involving the cancer cells, the associated stromal microenvironment and distant normal host cells, mostly from the bone marrow. Indeed, intricate tumor-host interactions are increasingly recognized as critical features of cancer. In particular, infiltrating cells of the immune system are crucial constituents of tumors and an important source of the growth stimulatory signals to the tumor cells. Tumor cells are surrounded by stromal cells, such as fibroblasts, lymphocytes, neutrophils, macrophages and mast cells, which communicate via a complex network of intercellular signaling pathways, mediated by surface adhesion molecules, cytokines and their receptors. However, the possible roles of these cells and molecules in the maintenance of micro-tumors in an occult state and in the induction of exit from the dormant state are not fully elucidated. In this review, we summarize recent findings and the current understanding of the role of bone marrow-derived cells, their recruitment into tumors and their interactive crosstalk with tumor cells, in leading to either the maintenance of, or exit from, tumor dormancy. Understanding the mechanisms of tumor growth and metastatic recurrence after periods of indolence is crucial for improving early detection, as well as increasing the cure rate for cancer patients.
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