Dynamics of metastasis suppressor gene inactivation

For most cancer cell types, the acquisition of metastatic ability leads to clinically incurable disease. Twelve metastasis suppressor genes (MSGs) have been identified that reduce the metastatic propensity of cancer cells. If these genes are inactivated in both alleles, metastatic ability is promoted. Here, we develop a mathematical model of the dynamics of MSG inactivation and calculate the expected number of metastases formed by a tumor. We analyse the effects of increased mutation rates and different fitness values of cells with one or two inactivated alleles on the ability of a tumor to form metastases. We find that mutations that are negatively selected in the main tumor are unlikely to be responsible for the majority of metastases produced by a tumor. Most metastases-causing mutations will be present in all (or most) cells in the main tumor.     

Mesenchymal transition and dissemination of cancer cells is driven by myeloid-derived suppressor cells infiltrating the primary tumor

In order to metastasize, cancer cells need to acquire a motile phenotype. Previously, development of this phenotype was thought to rely on the acquisition of selected, random mutations and thus would occur late in cancer progression. However, recent studies show that cancer cells disseminate early, implying the existence of a different, faster route to the metastatic motile phenotype. Using a spontaneous murine model of melanoma, we show that a subset of bone marrow-derived immune cells (myeloid-derived suppressor cells or MDSC) preferentially infiltrates the primary tumor and actively promotes cancer cell dissemination by inducing epithelial-mesenchymal transition (EMT). CXCL5 is the main chemokine attracting MDSC to the primary tumor. In vitro assay using purified MDSC showed that TGF-β, EGF, and HGF signaling pathways are all used by MDSC to induce EMT in cancer cells. These findings explain how cancer cells acquire a motile phenotype so early and provide a mechanistic explanation for the long recognized link between inflammation and cancer progression.     

Linear Model of Colon Cancer Initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

Linear model of colon cancer initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

Systemic Cancer Progression and Tumor Dormancy: Mathematical Models Meet Single Cell Genomics

Metastatic progression is thought to result from genetically advanced ?fully-malignant“ tumor cells. Within the concept the prevailing view holds that such cells disseminate mostly from large tumors and are capable of growing into metastases once they arrive at a distant site. Support for this scenario comes from numerous mouse models in which transplanted tumor cells grow into metastases within days or weeks. However, the assumption of such fully-malignant disseminating cells in human cancer is misleading and is neither supported by mathematical modeling of survival data from cancer patients nor by ex-vivo genomic data from disseminated cancer cells. For example, in breast cancer the growth of metastases is highly homogeneous and takes on average six years, the number of disseminated tumor cells before diagnosis of metastasis is similar for different tumor stages, and the genomic aberrations of disseminated cancer cells do rarely correspond to those in the primary tumor. Since these facts question conventional concepts of metastatic progression we provide a model of cancer progression in which time considerations and direct ex-vivo data form a starting point. In the proposed model tumor dormancy is a characteristic of almost all migrated tumor cells and metastatic growth is a rare, stochastic, evolutionary process of selection and mutation of cells that often disseminate shortly after transformation at the primary site.     

Reversible regulation of metastasis by ROS-generating mtDNA mutations

It has been controversial whether mtDNA mutations are responsible for oncogenic transformation (normal cells to develop tumors), and for malignant progression (tumor cells to develop metastases). To clarify this issue, we created trans-mitochondrial cybrids with mtDNA exchanged between mouse tumor cells that express different metastatic phenotypes. The G13997A mutation in the ND6 gene of mtDNA from high metastatic tumor cells reversibly controlled development of metastases by overproduction of reactive oxygen species (ROS), but did not control development of tumors. The mtDNA-mediated reversible control of metastasis reveals a novel function of mtDNA, and suggests that ROS scavengers may be therapeutically effective in suppressing metastasis.     

Correlation between Plasma DNA and Tumor Status in an Animal Model

Overcoming metastasis is one of the most important issues with lung cancer. Since metastasis arises through complex steps, a suitable animal model is indispensable for investigation of metastasis. To establish an animal model reflecting human metastatic lung cancers, we used NOD/SCID/Jak3^null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and dendritic cell function, and complement activation, as well as T and B cell deficiencies. After screening twenty human lung cancer cell lines through expression patterns of E-cadherin and vimentin according to epithelial mesenchymal transition features, an H1975 cell line carrying EGFR mutations, L858R and T790M, was selected for investigation. Inoculation of the cells into the dorsal flanks caused systemic metastases after one month in lymph nodes, liver, lung, and peritoneum, suggesting that metastases occurred both lymphogenically and hematogenously. We confirmed the existence of H1975 cells in metastatic lesions by detection of T790M and L858R using the mutation-biased PCR and quenching probe (MBP-QP) system previously established in our laboratory. In addition, tumor-derived plasma DNA could be detected using the MBP-QP method. The amount of tumor-derived DNA was associated with tumor volume, whereas an unrelated large amount of tumor-derived DNA was circulating in the presence of metastasis. We present a novel animal model with systemic metastasis with human lung cancer cells. The amount of tumor derived DNA would be related with tumor volume and tumor progression such as metastasis.     

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.     

Mutational signatures of de-differentiation in functional non-coding regions of melanoma genomes

Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer.     

Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers

During cancer progression, malignant cells undergo epithelial-mesenchymal transitions (EMT) and mesenchymal-epithelial transitions (MET) as part of a broad invasion and metastasis program. We previously observed MET events among lung metastases in a preclinical model of prostate adenocarcinoma that suggested a relationship between epithelial plasticity and metastatic spread. We thus sought to translate these findings into clinical evidence by examining the existence of EMT in circulating tumor cells (CTC) from patients with progressive metastatic solid tumors, with a focus on men with castration-resistant prostate cancer (CRPC) and women with metastatic breast cancer. We showed that the majority (> 80%) of these CTCs in patients with metastatic CRPC coexpress epithelial proteins such as epithelial cell adhesion molecule (EpCAM), cytokeratins (CK), and E-cadherin, with mesenchymal proteins including vimentin, N-cadherin and O-cadherin, and the stem cell marker CD133. Equally, we found that more than 75% of CTCs from women with metastatic breast cancer coexpress CK, vimentin, and N-cadherin. The existence and high frequency of these CTCs coexpressing epithelial, mesenchymal, and stem cell markers in patients with progressive metastases has important implications for the application and interpretation of approved methods to detect CTCs.     

Pleiotropic activity of lysophosphatidic acid in bone metastasis

Bone is a common metastatic site for solid cancers. Bone homeostasis is tightly regulated by intimate cross-talks between osteoblast (bone forming cells) and osteoclasts (bone resorbing cells). Once in the bone microenvironment, metastatic cells do not alter bone directly but instead perturb the physiological balance of the bone remodeling process controlled by bone cells. Tumor cells produce growth factors and cytokines stimulating either osteoclast activity leading to osteolytic lesions or osteoblast function resulting in osteoblastic metastases. Growth factors, released from the resorbed bone matrix or throughout osteoblastic bone formation, sustain tumor growth. Therefore, bone metastases are the sites of vicious cycles wherein tumor growth and bone metabolism sustain each other. Lysophosphatidic acid (LPA) promotes the growth of primary tumors and metastatic dissemination of cancer cells. We have shown that by acting on cancer cells via the contribution of blood platelets and the LPA-producing enzyme Autotaxin (ATX), LPA promotes the progression of osteolytic bone metastases in animal models. In the light of recent reports it would appear that the role of LPA in the context of bone metastases is complex involving multiple sources of lipid combined with direct and indirect effects on target cells. This review will present our current knowledge on the LPA/ATX axis involvement in osteolytic and osteoblastic skeletal metastases and will discuss the potential activity of LPA upstream and downstream metastasis seeding of cancer cells to bone as well as its implication in cancer induced bone pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Multiorgan metastasis of human HER-2⁺ breast cancer in Rag2⁻/⁻;Il2rg⁻/⁻ mice and treatment with PI3K inhibitor

In vivo studies of the metastatic process are severely hampered by the fact that most human tumor cell lines derived from highly metastatic tumors fail to consistently metastasize in immunodeficient mice like nude mice. We describe a model system based on a highly immunodeficient double knockout mouse, Rag2?/?;Il2rg?/?, which lacks T, B and NK cell activity. In this model human metastatic HER-2? breast cancer cells displayed their full multiorgan metastatic potential, without the need for selections or additional manipulations of the system. Human HER-2? breast cancer cell lines MDA-MB-453 and BT-474 injected into Rag2?/?;Il2rg?/? mice faithfully reproduced human cancer dissemination, with multiple metastatic sites that included lungs, bones, brain, liver, ovaries, and others. Multiorgan metastatic spread was obtained both from local tumors, growing orthotopically or subcutaneously, and from cells injected intravenously. The problem of brain recurrencies is acutely felt in HER-2? breast cancer, because monoclonal antibodies against HER-2 penetrate poorly the blood-brain barrier. We studied whether a novel oral small molecule inhibitor of downstream PI3K, selected for its penetration of the blood-brain barrier, could affect multiorgan metastatic spread in Rag2?/?; Il2rg?/? mice. NVP-BKM120 effectively controlled metastatic growth in multiple organs, and resulted in a significant proportion of mice free from brain and bone metastases. Human HER-2? human breast cancer cells in Rag2?/?;Il2rg?/? mice faithfully reproduced the multiorgan metastatic pattern observed in patients, thus allowing the investigation of metastatic mechanisms and the preclinical study of novel antimetastatic agents.     

Immunotherapy eradicates metastases with reversible defects in MHC class I expression

Tumor or metastatic cells lose MHC class I (MHC-I) expression during cancer progression as an escape mechanism from immune surveillance. These defects in MHC-I may be reversible by cytokines or different agents (soft lesions) or irreversible due to structural defects (hard lesions). The nature of these MHC-I alterations might determine the success or failure of immunotherapy treatments. In this study, we have used an MHC-I-positive murine fibrosarcoma tumor clone, GR9-A7, which generates multiple lung and lymph node metastases with reversible MHC-I alterations after treatment with IFN-γ. Four different antitumor treatments were carried out after primary tumor excision to determine their capacity to inhibit spontaneous metastatic colonization of the GR9-A7 tumor clone. We found that 2 different immunotherapy protocols (CpG plus autologous irradiated-GR9-A7 cells and protein-bound polysaccharide K (PSK) and 1 chemoimmunotherapy (docetaxel plus PSK) induced eradication of metastases. In contrast, chemotherapy with docetaxel alone produced only partial reduction in the number of metastases. Flow cytometric analysis of lymphocyte populations showed an immunosuppression in GR9-A7 tumor-bearing host, which could be reverted by immunotherapy treatments. Our results suggest that irreversible or reversible MHC-I alterations in tumor target cells may determine its progression or regression independently of the type of immunotherapy used.     

Detection of circulating tumor cells in colorectal cancer by immunobead-PCR is a sensitive prognostic marker for relapse of disease.

BACKGROUND: Recurrent and metastatic carcinoma of the colorectum remains a major problem, with survival at 5 years post curative resection still only about 50%. Moreover, up to 30% of patients who present with early stage disease also relapse and die within 5 years, suggesting the presence of micrometastatic disease at diagnosis. One route of metastatic spread is via the blood stream, hence the detection of tumor cells in blood is likely to provide an important predictive tool with respect to relapse of disease. We have developed a sensitive molecular technique to identify tumor cells in blood using mutations in codon 12 of the K-ras gene as a marker. MATERIALS AND METHODS: Twenty-seven patients whose tumor carried a mutation in codon 12 of K-ras were studied for the presence of tumor cells in perioperative peripheral blood samples. Immunomagnetic beads, labeled with an epithelial-specific antibody, were used to harvest epithelial cells from blood. K-ras mutations were identified in this selected population using a polymerase chain reaction (PCR)-based analysis (immunobead-PCR). RESULTS: Circulating K-ras mutant cells were detected in 9 or 27 patients; seven of these nine patients have since died due to recurrent or metastatic disease. Mutant cells were not detected in 18 patients, and 16 or 18 have remained disease free (median follow-up: 16 months; range: 7-42 months). Kaplan-Meier analysis showed that detection of K-ras mutant cells in bloods was associated with significantly reduced disease-free survival (p = 0.0001). CONCLUSION: This study indicates that detection of circulating tumor cells perioperatively by immunobead-PCR provides a sensitive prognostic marker for recurrent and metastatic colorectal cancer.     

A mouse model of pulmonary metastasis from spontaneous osteosarcoma monitored in vivo by Luciferase imaging

Background

Osteosarcoma (OSA) is lethal when metastatic after chemotherapy and/or surgical treatment. Thus animal models are necessary to study the OSA metastatic spread and to validate novel therapies able to control the systemic disease. We report the development of a syngeneic (Balb/c) murine OSA model, using a cell line derived from a spontaneous murine tumor.

Methodology

The tumorigenic and metastatic ability of OSA cell lines were assayed after orthotopic injection in mice distal femur. Expression profiling was carried out to characterize the parental and metastatic cell lines. Cells from metastases were propagated and engineered to express Luciferase, in order to follow metastases in vivo.

Principal Findings

Luciferase bioluminescence allowed to monitor the primary tumor growth and revealed the appearance of spontaneous pulmonary metastases. In vivo assays showed that metastasis is a stable property of metastatic OSA cell lines after both propagation in culture and luciferase trasduction. When compared to parental cell line, both unmodified and genetically marked metastatic cells, showed comparable and stable differential expression of the enpp4, pfn2 and prkcd genes, already associated to the metastatic phenotype in human cancer.

Conclusions

This OSA animal model faithfully recapitulates some of the most important features of the human malignancy, such as lung metastatization. Moreover, the non-invasive imaging allows monitoring the tumor progression in living mice. A great asset of this model is the metastatic phenotype, which is a stable property, not modifiable after genetic manipulation.     

Angiotensin II facilitates breast cancer cell migration and metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.     

Data on the Recurrence of Breast Tumors Fit a Model in which Dormant Cells are Subject to Slow Attrition but Can Randomly Awaken to Become Malignant

We successfully modeled the recurrence of tumors in breast cancer patients, assuming that:(i) A breast cancer patient is likely to have some circulating metastatic cells, even after initial surgery. (ii) These metastatic cells are dormant. (iii) The dormant cells are subject to attrition by the body’s immune system, or by random apoptosis or senescence.(iv) Recurrence suppressor mechanisms exist. (v) When such genes are disabled by random mutations, the dormant metastatic cell is activated, and will develop to a cancer recurrence. The model was also fitted to data on the survival of pancreatic cancer patients. The time course of cancer recurrence in a group of poor prognosis breast cancer patients could not be linked to the over- (or under-) expression of any gene in the primary tumors from which the recurrent tumors derived. Thus, the recurrence of the tumor in breast cancer patients appears to be a random event. Inasmuch as the kinetics of cancer recurrence in published data sets closely follows the model found for the appearance of sporadic retinoblastoma, tumor recurrence could be triggered by mutations in awakening-suppressor mechanisms. The retinoblastoma tumor suppressor gene was identified by tracing its occurrence in familial retinoblastoma pedigrees. Will it be possible to track the postulated cancer recurrence, awakening suppressor gene(s) in early recurrence breast cancer patients?