Faculty Bibliography

Tumor necrosis factor alpha (TNF alpha) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNF alpha, the participation of TNF alpha receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNFalpha induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappa B (NF-kappa B) transcriptional activation. A TNF alpha-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-kappa B transcriptional activation and cell proliferation, just like wild-type TNF alpha, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNF alpha signaling and biological effect. Moreover, in vivo TNF alpha administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-kappa B activity, Bay 11-7082, resulted in regression of TNF alpha-promoted tumor. Bay 11-7082 blocked TNF alpha capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-xLin vivo and in vitro. Our results reveal evidence for TNF alpha as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNF alpha antagonists and NF-kappa B pharmacological inhibitors in established breast cancer treatment.

Progestin regulation of gene expression was assessed in the progestin-dependent murine tumor line C4HD which requires MPA, a synthetic progestin, for in vivo growth and expresses high levels of progesterone receptor (PR). By using suppressive subtractive hybridization, caveolin-1 was identified as a gene whose expression was increased with in vivo MPA treatment. By Northern and Western blot analysis, we further confirmed that caveolin-1 mRNA and protein expression increased in MPA-treated tumors as compared with untreated tumors. When primary cultures of C4HD cells were treated in vitro with MPA, caveolin-1 levels also increased, effect that was abolished by pre-treatment with progestin antagonist RU486. In addition, MPA promoted strong caveolin-1 promoter transcriptional activation both in mouse and human breast cancer cells. We also showed that MPA regulation of caveolin-1 expression involved in activation of two signaling pathways: MAPK and PI-3K. Short-term MPA treatment of C4HD cells led to tyrosine phosphorylation of caveolin-1 protein, where Src was the kinase involved. Additionally, we showed that MPA-induced association of caveolin-1 and PR, which was detected by coimmunoprecipitation and by confocal microscopy. Finally, we proved that MPA-induced proliferation of C4HD cells was inhibited by suppression of caveolin-1 expression with antisense oligodeoxynucleotides to caveolin-1 mRNA. Furthermore, we observed that inhibition of caveolin-1 expression abrogated PR capacity to induced luciferase activity from a progesterone response element-driven reporter plasmid. Comprehensively, our results demonstrated for the first time that caveolin-1 expression is upregulated by progestin in breast cancer. We also demonstrated that caveolin-1 is a downstream effector of MPA that is partially responsible for the stimulation of growth of breast cancer cells.