Increased sensitivity of African American triple negative breast cancer cells to nitric oxide-induced mitochondria-mediated apoptosis.

BackgroundBreast cancer is a complex heterogeneous disease where many distinct subtypes are found. Younger African American (AA) women often present themselves with aggressive form of breast cancer with unique biology which is very difficult to treat. Better understanding the biology of AA breast tumors could lead to development of effective treatment strategies. Our previous studies indicate that AA but not Caucasian (CA) triple negative (TN) breast cancer cells were sensitive to nitrosative stress-induced cell death. In this study, we elucidate possible mechanisms that contribute to nitric oxide (NO)-induced apoptosis in AA TN breast cancer cells.MethodsBreast cancer cells were treated with various concentrations of long-acting NO donor, DETA-NONOate and cell viability was determined by trypan blue exclusion assay. Apoptosis was determined by TUNEL and caspase 3 activity as well as changes in mitochondrial membrane potential. Caspase 3 and Bax cleavage, levels of Cu/Zn superoxide dismutase (SOD) and Mn SOD was assessed by immunoblot analysis. Inhibition of Bax cleavage by Calpain inhibitor, and levels of reactive oxygen species (ROS) as well as SOD activity was measured in NO-induced apoptosis. In vitro and in vivo effect of NO treatment on mammary cancer stem cells (MCSCs) was assessed.Results and discussionNO induced mitocondria-mediated apoptosis in all AA but not in CA TN breast cancer cells. We found significant TUNEL-positive cells, cleavage of Bax and caspase-3 activation as well as depolarization mitochondrial membrane potential only in AA TN breast cancer cells exposed to NO. Inhibition of Bax cleavage and quenching of ROS partially inhibited NO-induced apoptosis in AA TN cells. Increase in ROS coincided with reduction in SOD activity in AA TN breast cancer cells. Furthermore, NO treatment of AA TN breast cancer cells dramatically reduced aldehyde dehydrogenase1 (ALDH1) expressing MCSCs and xenograft formation but not in breast cancer cells from CA origin.ConclusionsEthnic differences in breast tumors dictate a need for tailoring treatment options more suited to the unique biology of the disease

Induction of DUSP9 in Xenografts from Human Breast Cancer Cell Lines increases Mammary Cancer Stem Cells

Breast cancer remains a complex disease that kills 40,000 women every year. Initiation and progression of breast cancer is influenced by heterogeneous groups of cells, including mammary cancer stem cells (MCSCs). Progression of this dreadful disease is driven by many signaling pathways among which MAPK pathway is highly prominent. Since targeting prominent kinases in MAPK pathway has been unsuccessful to control breast cancer, it is important to examine the phosphatases that regulate the activity of these kinases. Using xenograft model from breast cancer cell lines, our lab has found that during the initial stages of xenograft development (week 1-4, 100-200mg weight), ERK1/2 remains inactive. However, during the later stages of tumor development (week 5-15, 300-700mg weight), we found phospho-ERK1/2, which is the active form of ERK1/2 remains highly up regulated. Our lab has also found that presence of inactive ERK1/2 during the initial stages of tumor development was independent of pMEK1/2, which is the upstream kinase that activates ERK1/2. We have also found that a specific dual specific phosphatase (DUSP9) was induced in the xenografts from breast cancer cell lines during initial stages of their development. I will be examining the significance of DUSP9 induction in the early stages of xenograft development. There are reports that DUSP9-mediated pERK1/2 inactivation has been found to increase mouse embryonic stem cell content. I have been examining the expression levels of MCSCS (Aldehyde dehydrogenase 1(ALDH1)/ OCT3/4 / CD44/SOX2) in various stages of xenograft development and correlating them with DUSP9 expression and pERK1/2. I am examining the expression levels of MCSCs by Western blot analysis as well as qPCR. I have also been treating breast cancer cell line HMLEHRASV12 with MEK inhibitors, in vitro, for inactivating ERK to analyze the levels of mammary cancer stem cells (ALDH1)/ OCT3/4 / CD44/SOX2. I am also examining the levels of DUSP9 as well as the levels of ERK/pERK1/2 /AKT /pAKT in cells treated with MEK1/2 inhibitors in vitro. I intend to determine whether inhibition of pERK1/2 could influence embryonic stem cell content as analyzed by the expression of markers Oct ¾ and Sox2

COX2 Plays an Important Role in Maintenance of Mammary Cancer Stem Cell Population in Human Breast Cancer Cell Lines

Breast cancer is a malignant growth in the adipose-rich mammary gland. Initiation and progression of breast tumors involve multiple cell types, among which mammary cancer stem cells play an important role. There are two main types of adipocytes; white adipocytes have previously been found to influence the disease while brown adipocytes, which uniquely express uncoupling protein 1 (UCP1), were recently detected in breast tumors. We have detected Myf5, a transcription factor, which is expressed in the progenitor population of brown adipocytes, in breast cancer cell lines. We also found an association between expression of Myf5 and COX2 (or Ptgs2), which induces inflammation. We hypothesize that inflammatory conditions induced by COX2 could be contributing to the maintenance as well as expansion of Myf5 progenitor population. Breast cancer cell lines (HCC1806, HCC 70 and MDA-MB-468) were treated with various concentrations of Cox2-inhibitor (SC 236) and Cox1-inhibitor (SC 560) for 24 hours. They were analyzed for the expression of Myf5 and mammary cancer stem cell markers (ALDH1, CD44, Oct 3/4), by Western blot and quantitative Polymerase Chain Reaction (qPCR). We also examined the levels of inflammatory markers (Cox1 and Cox2) and various inducers of COX2 by Western blot analysis and qPCR. Our preliminary results suggest that breast cancer cells treated specifically COX2 inhibitor exhibit a decline in Myf5, ALDH1, and CD44 and an increase in protein levels of COX2. This suggests that COX2 might play a role in maintenance of mammary cancer stem cells, including Myf5 progenitor population in human breast cancer cell lines

Increased sensitivity of African American triple negative breast cancer cells to nitric oxide-induced mitochondria-mediated apoptosis.

BackgroundBreast cancer is a complex heterogeneous disease where many distinct subtypes are found. Younger African American (AA) women often present themselves with aggressive form of breast cancer with unique biology which is very difficult to treat. Better understanding the biology of AA breast tumors could lead to development of effective treatment strategies. Our previous studies indicate that AA but not Caucasian (CA) triple negative (TN) breast cancer cells were sensitive to nitrosative stress-induced cell death. In this study, we elucidate possible mechanisms that contribute to nitric oxide (NO)-induced apoptosis in AA TN breast cancer cells.MethodsBreast cancer cells were treated with various concentrations of long-acting NO donor, DETA-NONOate and cell viability was determined by trypan blue exclusion assay. Apoptosis was determined by TUNEL and caspase 3 activity as well as changes in mitochondrial membrane potential. Caspase 3 and Bax cleavage, levels of Cu/Zn superoxide dismutase (SOD) and Mn SOD was assessed by immunoblot analysis. Inhibition of Bax cleavage by Calpain inhibitor, and levels of reactive oxygen species (ROS) as well as SOD activity was measured in NO-induced apoptosis. In vitro and in vivo effect of NO treatment on mammary cancer stem cells (MCSCs) was assessed.Results and discussionNO induced mitocondria-mediated apoptosis in all AA but not in CA TN breast cancer cells. We found significant TUNEL-positive cells, cleavage of Bax and caspase-3 activation as well as depolarization mitochondrial membrane potential only in AA TN breast cancer cells exposed to NO. Inhibition of Bax cleavage and quenching of ROS partially inhibited NO-induced apoptosis in AA TN cells. Increase in ROS coincided with reduction in SOD activity in AA TN breast cancer cells. Furthermore, NO treatment of AA TN breast cancer cells dramatically reduced aldehyde dehydrogenase1 (ALDH1) expressing MCSCs and xenograft formation but not in breast cancer cells from CA origin.ConclusionsEthnic differences in breast tumors dictate a need for tailoring treatment options more suited to the unique biology of the disease

Increased Expression of Beige/Brown Adipose Markers from Host and Breast Cancer Cells Influence Xenograft Formation in Mice

The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment (TME). 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, were 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 (MCSCs) that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1(+) or Myf5(+) cells significantly reduced tumor development. There was increased COX-2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX-2 inhibitor (SC236) reduced tumor growth. By 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 (BAT)-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