Tumor necrosis factor alpha promotes in vivo growth of a murine mammary adenocarcinoma

MARTíN A. RIVAS, MERCEDES TKACH, ROMINA P. CARNEVALE, CINTHIA ROSEMBLIT, CECILIA J. PROIETTI, WENDY BEGUELIN, ISABEL FRAHM, EDUARDO H. CHARREAU, PATRICIA V. ELIZALDE, ROXANA SCHILLACI.

San Diego, USA

Congreso; AACR Annual Meeting; 2008

American Associatiom of Cancer Research (AACR)

We have previously shown that tumor necrosis factor alpha (TNFá) induces in vitro proliferation of the murine mammary adenocarcinoma C4HD, activating signaling pathways such as PI3-K/Akt, that converge on the transcriptional activation of NF-êB. C4HD cells belong to an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice. The aim of the present work was to investigate TNFá ability to support C4HD tumor in vivo growth and to examine a possible cross-talk with progestins. Mice were inoculated subcutaneously (s.c.) into the left flank of mice with a fragment of C4HD tumor (1mm3) and were injected either s.c. with 0.5 ìg/kg mTNFá/day or with PBS near the inoculation site. Tumor incidence at day 15 was 76% (n=26) for the TNFá-treated group, while no mice from the control group (PBS) developed the tumor. When tumors reached a mean volume of 60-70 mm3, animals were randomly assigned into four groups and treated as follows: PBS; 0.5 ìg/kg mTNFá/day; 0.5 ìg/kg mTNFá/day + 5 mg/kg Bay 11-7082, a NF-êB inhibitor, i.p./3 days; or 0.5 ìg/kg mTNFá/day+ i.p. vehicle/3 days. Administration of Bay 11-7082 proved effective to inhibit both tumor growth rate and tumor volume relative to TNFá or TNFá plus vehicle-treated animals. Histological examination determined a decrease in mitotic index and a striking increase in fibrosis. Moreover, tumor significantly regressed in the animals assigned to PBS treatment, evidencing a TNFá-dependent growth pattern. By immunohistochemistry in tumor sections, we confirmed that NF-êB subunit p65 remained cytoplasmatic in the presence of Bay 11-7082, thus showing its effectively, while no changes in phospho-Ser 473-Akt staining were found. Interestingly, all these data demonstrated that TNFá is able to support C4HD growth, a tumor which was previously reported to be progestin-dependent. We then studied the possible interaction between TNFá and progestin. We found that simultaneous in vivo administration of TNFá and MPA 40 mg/mice enhanced C4HD tumor growth as compared with each treatment alone. Moreover, in primary culture of C4HD cells the presence of TNFá and MPA produced an additive effect on cell proliferation. In order to find out the possible mechanism mediating this effect, we studied progesterone receptor binding in vitro by using the single-saturating-dose 3H R5020 binding assay, and observed that TNFá was able to upregulate progesterone-binding sites in C4HD cells at 48h of treatment. Our results are the first evidence of TNFá as an in vivo tumor promoter in breast cancer, and also for the first time reveal that Bay 11-7082 may be used as a chemotherapeutic agent in the treatment of an established tumor. Finally, we here describe a previously unknown interaction between TNFá and progestin in breast cancer tumor growth.