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Lactoferrin induces TIMP-4 mediated anti-apoptosis in breast cancer Open Access

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Breast cancer makes up 28% of all cancers affecting women with 1 in 8 in the US developing invasive forms. Here we examine the effects of Lactoferrin- a transcription factor secreted in breast milk, on the activity of TIMP-4, a member of the family of tissue inhibitor of metalloproteinases (TIMPs). TIMPs regulate the extracellular matrix (ECM) and anti-apoptotic signaling pathway. Recent findings from our lab showed that treatment of non-TNBC cell lines with lactoferrin can render them a triple-negative phenotype and promote invasiveness. Also, imbalance in the homeostatic proportions of matrix metalloproteinases and TIMPs facilitates ECM degradation and cell migration and invasion. TIMP-4 has been reported to have significant association with poor patient prognosis and survival in early stage breast carcinomas. We hypothesize that Lactoferrin in conjunction with TIMP-4 may have a role in contributing to the mechanism of cell survival and by implication, in invasiveness. To determine whether lactoferrin can affect TIMP-4 expression levels, we probed for known lactoferrin (Lf) consensus sequences in the promoter region of TIMP-4. Next, we aimed to confirm such interaction by electrophoretic mobility shift assays and chromatin immunoprecipitation. We also investigated the regulation of TIMP-4 in lactoferrin treated cell lines by q-PCR and luciferase reporter assay and examined the levels of secreted TIMP-4 and its downstream targets in breast cancer cells. We identified three putative lactoferrin binding sites in the promoter region of TIMPs. TIMP-4 had the best match for consensus sequences which was further verified by EMSA. We also confirmed significant up-regulation of TIMP-4 expression by Q-PCR and luciferase reporter activity in lactoferrin treated breast cancer lines. Immunoblotting further revealed induction of secreted TIMP-4 over various time-points. Signaling for cell survival was confirmed by stimulation of the PI3K/Akt anti-apoptotic pathway. Our studies show that Lactoferrin up-regulates TIMP-4 expression and induces its secretion in breast cancer cell lines, possibly upregulating the PI3K/Akt anti-apoptotic signaling signifying the importance of this particular cell survival pathway. Moreover, as lactoferrin initiates TIMP-4 mediated signaling, it calls for further investigation into the contribution of lactoferrin and TIMP-4, both independently and in conjunction, in regulating cell survival and tumor progression.

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