Articles by Michael M. Lizardo in JoVE
Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay Michael M. Lizardo1,2, Poul H. Sorensen2,3 1Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2BC Cancer Agency, Provincial Health Services Authority, 3Department of Pathology and Laboratory Medicine, University of British Columbia The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.
Other articles by Michael M. Lizardo on PubMed
Tumor Microenvironment-based Feed-forward Regulation of NOS2 in Breast Cancer Progression Proceedings of the National Academy of Sciences of the United States of America. | Pubmed ID: 24733928 Inflammation is widely recognized as an inducer of cancer progression. The inflammation-associated enzyme, inducible nitric oxide synthase (NOS2), has emerged as a candidate oncogene in estrogen receptor (ER)-negative breast cancer, and its increased expression is associated with disease aggressiveness and poor survival. Although these observations implicate NOS2 as an attractive therapeutic target, the mechanisms of both NOS2 induction in tumors and nitric oxide (NO)-driven cancer progression are not fully understood. To enhance our mechanistic understanding of NOS2 induction in tumors and its role in tumor biology, we used stimulants of NOS2 expression in ER(-) and ER(+) breast cancer cells and examined downstream NO-dependent effects. Herein, we show that up-regulation of NOS2 occurs in response to hypoxia, serum withdrawal, IFN-γ, and exogenous NO, consistent with a feed-forward regulation of NO production by the tumor microenvironment in breast cancer biology. Moreover, we found that key indicators of an aggressive cancer phenotype including increased S100 calcium binding protein A8, IL-6, IL-8, and tissue inhibitor matrix metalloproteinase-1 are up-regulated by these NOS2 stimulants, whereas inhibition of NOS2 in MDA-MB-231 breast cancer cells suppressed these markers. Moreover, NO altered cellular migration and chemoresistance of MDA-MB-231 cells to Taxol. Most notably, MDA-MB-231 tumor xenographs and cell metastases from the fat pad to the brain were significantly suppressed by NOS2 inhibition in nude mice. In summary, these results link elevated NOS2 to signals from the tumor microenvironment that arise with cancer progression and show that NO production regulates chemoresistance and metastasis of breast cancer cells.
Upregulation of Glucose-Regulated Protein 78 in Metastatic Cancer Cells Is Necessary for Lung Metastasis Progression Neoplasia (New York, N.Y.). | Pubmed ID: 27973325 Metastasis is the cause of more than 90% of all cancer deaths. Despite this fact, most anticancer therapeutics currently in clinical use have limited efficacy in treating established metastases. Here, we identify the endoplasmic reticulum chaperone protein, glucose-regulated protein 78 (GRP78), as a metastatic dependency in several highly metastatic cancer cell models. We find that GRP78 is consistently upregulated when highly metastatic cancer cells colonize the lung microenvironment and that mitigation of GRP78 upregulation via short hairpin RNA or treatment with the small molecule IT-139, which is currently under clinical investigation for the treatment of primary tumors, inhibits metastatic growth in the lung microenvironment. Inhibition of GRP78 upregulation and an associated reduction in metastatic potential have been shown in four highly metastatic cell line models: three human osteosarcomas and one murine mammary adenocarcinoma. Lastly, we show that downmodulation of GRP78 in highly metastatic cancer cells significantly increases median survival times in our in vivo animal model of experimental metastasis. Collectively, our data indicate that GRP78 is an attractive target for the development of antimetastatic therapies.
Positively Selected Enhancer Elements Endow Osteosarcoma Cells with Metastatic Competence Nature Medicine. | Pubmed ID: 29334376 Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.