The mechanism of TNF-stimulated growth of mammary epithelial cells: evidence that NFκB2/p52 can compensate for NFκB1/p50

3649 Nuclear factor kappaB (NFκB) is important in mammary gland development and breast cancer. We previously demonstrated that TNF stimulated the growth of rat mammary epithelial cells (MEC) and rat mammary tumor cells in a physiologically relevant 3D primary culture system, accompanied by enhanced DNA-binding of the NFκB p50 homodimer. Recently, we found that expression of cyclin D1 mRNA and protein was also increased by TNF in MEC. To determine if NFκB1/p50 was required for TNF-stimulated growth and up-regulation of cyclin D1, the TNF responsiveness of MEC including cell growth, activation of NFκB and cyclin D1 expression was compared between NFκB1/p50 null and wild type mice in MEC primary culture. TNF stimulated growth and induced the expression of cyclin D1 in MEC from both p50 null and wild type mice. No p50 protein was detected in either mammary gland tissue or cultured MEC of p50 null mice. However, a TNF-inducible NFκB DNA-binding protein complex was seen in the nuclear extract of cultured MEC from p50 null mice by electrophoresis mobility shift assay. This complex migrated at the same position as the upper band of the two NFκB-binding complexes detected in wild type MEC. Supershift analysis showed that the NFκB-binding complex in p50 null MEC was composed of NFκB2/p52 and RelB, while the two complexes in the wild type mice were the p65/p50 heterodimer, and the p50 homodimer. Additionally, NFκB2/p52 as well as its precursor p100 was increased by TNF in whole cell lysates of MEC from both p50 null and wild type mice. These data provide the first evidence that in the absence of p50, its close homologue p52 can functionally compensate. Although we demonstrate that the p50 protein itself is not critical in TNF-stimulated proliferation and up-regulation of cyclin D1 in MEC, the similar sensitivity of MEC to TNF in p50 null and wild type mice and the substitution of p52 for p50 highly suggest that NFκB-mediated transactivation and transcription are important in MEC responsiveness to TNF. The data further suggest that therapeutic approaches that target the NFκB pathway will have to take into consideration the individual proteins that make up the NFκB complex and the partial redundancy of the protein components within the complex. Supported by NIH CA77656.