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Upregulation of the markers of beige/brown adipocytes were not dependent on site of cancer cell injection.

Human breast tumors used in this study were according to the stage/grade

The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment. Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1, and OPLAH) as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers was also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1+ or Myf5+ cells significantly reduced tumor development. There was increased COX2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX2 inhibitor (SC236) reduced tumor growth. In contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient tumor tissues were generated that expressed key brown adipose tissue–related markers and contained cells that morphologically resembled brown adipocytes.Implications: This is the first report demonstrating that beige/brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design. Mol Cancer Res; 14(1); 78–92. ©2015 AACR.

Human specific and mouse specific primers were validated using human pheochromocytoma-derived peri-adrenal fat and mouse brown adipose tissue respectively.

Images of xenografts, osmium stained sections, quantitation of lipids and immunohistochemistry.

Human breast tumor tissues were characterized based on ethnicity and age

Sequence of various mouse and human primers used in this study.

Sections of xenografts were analyzed for co-expression of collagen and adiponectin.

PDF File - 90K, Loss of SREBP signaling results in the uncoupling of saturated fatty acid biosynthesis from desaturase activity.

PDF File - 90K, Graphical representation of method used to determine the relative contribution of de novo cholesterol and long chain fatty acids synthesis to the total cellular pool.

PDF File - 92K, Gene set enrichment analysis on the ten most statistically significant down-regulated metabolic pathways in SCAP loss-of-function cells.

PDF File - 158K, Inhibition of lipogenic transcriptional program in various cancer cells.

PDF File - 118K, Legends for Supplementary Figures 1-8.

PDF File - 92K, Cholesterol content is unchanged in SREBP loss-of-function cells.

PDF File - 155K, The lipogenic program and growth of glioma cells is SCAP sensitive.

PDF File - 52K, Cholesterol content is unchanged in SREBP loss-of-function cells.

PDF File - 112K, Expression of mature SREBP1 alters the ratio of saturated to monounsaturated fatty acids.

The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment. Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1, and OPLAH) as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers was also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1+ or Myf5+ cells significantly reduced tumor development. There was increased COX2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX2 inhibitor (SC236) reduced tumor growth. In contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient tumor tissues were generated that expressed key brown adipose tissue–related markers and contained cells that morphologically resembled brown adipocytes. Implications: This is the first report demonstrating that beige/brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design. Mol Cancer Res; 14(1); 78–92. ©2015 AACR.

HOXB13 is a homeobox gene whose dysregulation has been implicated in cancer. We have previously shown that HOXB13 plays an oncogenic role in ovarian cancer by driving epithelial-to-mesenchymal transition (EMT); however the mechanism by which this occurs has not been established. Global metabolomics profiling revealed that overexpression of HOXB13 in genetically defined mouse ovarian cancer cell lines inhibits succinate dehydrogenase (SDH) activity. Targeted-knockdown of the SDH subunit SdhB by shRNA results in mislocalization of E-cadherin and a spindle-shaped morphology suggestive of EMT. Metabolomics and real-time flux analysis showed that knockdown of SdhB results in elevated levels of succinate as well as defective mitochondrial respiration. Despite the mitochondrial respiration defect, SdhB knockdown cells display increased anchorage-independent growth in soft agar and enhanced xenograft tumor formation. Elevated levels of succinate have been shown to inhibit α-ketoglutarate requiring enzymes such as the Jumanji C (JmjC)-domain containing H3K27 demethylases, UTX and JMJD3. Indeed, knockdown of SdhB results in a hypermethylated epigenome as evidenced by increased levels of H3K27me2 and H3K27me3. Both the epigenetic and EMT phenotypes can be recapitulated by pharmacological inhibition of UTX and JMJD3 in control cells. These data point to a mechanism wherein modulation of oncometabolite levels can affect the epigenome, thereby influencing cancer cell morphology and tumorigenic properties. Citation Format: Paul-Joseph Aspuria, Dong-Joo Cheon, Maricel Gozo, Brenda Salumbides, Laurent Vergnes, John Asara, Karen Reue, Elizabeth Kensicki, Beth Karlan, Sandra Orsulic. HOXB13 inhibition of succinate dehydrogenase leads to epithelial-to-mesenchymal transition in mouse ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1088. doi:10.1158/1538-7445.AM2013-1088

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbor a fused, branching mitochondrial network, with oxidative phosphorylation (OXPHOS) as the main energy source. A role for mitochondria in hPSC bioenergetics therefore remains uncertain. Here we show that hPSCs have functional respiratory complexes that consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis. To help maintain hPSC mitochondrial membrane potential, cell proliferation, and viability, glycolytic ATP is hydrolyzed by the mitochondrial F1F0ATP synthase. Uncoupling protein 2 (UCP2) regulates this unique pattern of hPSC energy metabolism while suppressing reactive oxygen species. Upon differentiation, UCP2 is repressed and cells shift from glycolysis and fatty acid oxidation to glucose oxidation dependency. Ectopic UCP2 expression during differentiation blocks this metabolic transition and blocks proper embryoid body formation. Combined, our data show that hPSC have distinct bioenergetics regulated by UCP2 and repression of UCP2 is required for metabolic reprogramming along differentiation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2011-4360