Role of hypoxia and nitric oxide in the malignant behavior of a mammary tumor composed by luminal and myoepithelial cells

KRASNAPOLSKI MARTIN ALEJANDRO; BAL DE KIER JOFFÉ ELISA; EIJÁN ANA MARIA

San Diego, California, USA

Congreso; 99th Annual Meeting of the American Association for Cancer Research (AACR); 2008

American Association for Cancer Research (AACR)

Hypoxia, a common phenomenon in the tumor microenvironment leads to the accumulation of the á subunit of the transcription factor Hypoxia Induced Factor (HIF-1á), and the inducible Nitric Oxide Sintase (iNOS) (enzyme that produces high levels of NO). Cobalt salts generate pseudohypoxia, a condition in which, even in normoxic conditions, there is detectable HIF-1 transcriptional activity. From the murine mammary adenocarcinoma M38, composed by malignant luminal and myoepithelial cells we obtained the following cell lines: LM38-LP (bicellular), LM38-HP (epithelioid) and LM38-D2 (myoepithelial clone). The two last ones display a diminished in vivo growth rate and metastatic capacity than the parental tumor, uncovering the importance of the population interactions in their behavior. The main objective was to analyze several parameters of M38-derived cell lines to dissect some of the biological and molecular bases that underlie the cooperation. ·   LM38-D2 cells migration was significantly higher (p<0.01) than LM38-LP and LM38-HP cells (490+/-45 ìm/24hs; 284+/-55 ìm/24hs; 265+/-40 ìm/24hs, respectively). ·   Collagen gel invasion was 60+/-18 for LM38-D2 and 20+/-6 AU for the other two cell lines. ·   Matrigel invasion assays in Transwell chambers showed equivalent results (LM38-D2: 60+/-18 cells/field, LM38-LP: 16+/-6 cells/field and LM38-HP 12+/-4 cells/field). ·   In pseudohypoxia, LM38-LP and LM38-HP significantly increased (p<0.05) NO production (from 153±64 to 337±67 and 138±99 to 273±60 nmol/106 cells) (VER QUE QUEDE SUPERINDICE), effect that was inhibited by 1400W (iNOS specific inhibitor). In LM38-D2, NO was undetectable both in hypoxia and normoxia. ·   uPA activity, analyzed by radial caseinolysis, was significantly enhanced by pseudohypoxia only in LM38-LP (35.51±13.58 vs 57.82±10.31) and LM38-HP cells (17.18±4.13 vs 43.74±13.14). ·   By WB we observed that LM38-LP and LM38-HP cell lines increased HIF-1á  after 30 min treatment with CoCl2 while iNOS expression was enhanced after 3h of hypoxia. These molecules were undetectable In LM38-D2 cells. ·   In order to evaluate the function of NO we treated cells for 24h with different concentrations of DETA/NONOate, a NO donor. At 500ìM the metabolic activity of LM38-D2 cells, an assessment of the actual cell number, showed a 30% increase over untreated cells, without affecting neither LM38-LP nor LM38-HP. Tumor progression is a complex process involving various sequential steps. Our results suggest that the malignant behavior of M38 tumor, composed by luminal and myoepithelial cells, may be explained by the following model. The hypoxic conditions normally established at the initial stages of tumor growth lead to an enhanced NO production by the luminal component (through HIF-1á and iNOS) which, in turn, might stimulate myoepithelial cell proliferation and proteinases secretion, that, in a three-dimensional context might act as an invasive forefront, generating gaps that could help luminal cells to escape from the primary tumor.