acase@tulane.edu / 1 / David M. Cunningham

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Here we studied the role of hypoxia and hypoxia-induced factors in the development of breast cancer brain metastasis by using ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. We detected hypoxia noninvasively by using a novel spectroscopic photoacoustic tomography technology (SPAT). Sprague-Dawley rats inoculated intracranially with ENU1564, a carcinogen-induced rat mammary adenocarcinoma cell line, were imaged with SPAT three weeks post inoculation. Proteins important for tumor angiogenesis and invasion were detected in hypoxic brain foci identified by SPAT and were elevated compared with control brain. We showed that HIF-1α, MMP-9, VEGF-A, and VEGFR2 (Fkl-1) protein and mRNA expression levels were higher (P < 0.05) in brain tumor tissues compared to normal brain. We also found an increased expression of HIF-1α proteins, MMP-9, VEGF-A and VEGFR2 mRNA and proteins in hypoxic ENU1564 cells in vitro. We also demonstrated the involvement of PI3K-Akt pathway in hypoxic regulation of MMP-9 and VEGF but not VEGFR2 by using specific PI3K inhibitor. Using MEK1/2 inhibitor we showed that hypoxic regulation of MMP-9, VEGF-A and VEGFR2 also involve MEK1/2-ERK pathway. We also investigated the effect of fibroblast growth factor-1 (FGF-1), one of the factors known to be upregulated by hypoxia, on the expression of MMP-9 in ENU1564 cell line. We observed that FGF-1 induces an increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. Next, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-κBp65, and enhances degradation of cytoplasmic IκBα. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data showed that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-κB and AP-1 in FGF-1-treated cells and that mutation of either NF-κB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1.

Thesis (Ph. D.)--University of Hong Kong, 2008.

Inaug.-Diss.--Tübingen. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Inaug.-Diss.--Tübingen. / Vita. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The correlation of information obtained from in vitro investigations and in vivo experiments has frequently evaded researchers, especially in the area of tumor cell invasion and metastasis. In order to better understand and associate in vitro tumor cell invasion through basement membranes with in vivo tumor metastasis in syngeneic animal models, and the subsequent modulation of these processes, the following studies have been undertaken. Malignant murine melanoma cell lines designated B16F1 and B16F10, syngeneic to the C57BL6 mouse, a melanotic variant of the Cloudman S-91 melanoma cell line (denoted Mel-11a) with the syngeneic host being the DBA/2J mouse, and a malignant human melanoma line referenced as A375P (parental) and A375M (metastatic) were used for this dissertation project. Tumor cells were labeled with either ¹⁴C-thymidine or ¹²⁵I-deoxyuridine using previously established protocols. Radiolabeled tumor cells were introduced into the Membrane Invasion Culture System (MICS) in vitro, a system developed in our lab, and concomitantly into the lateral tail vein by injection or intracutaneously into the appropriate syngeneic host in the presence or absence of such biological response modifying agents as [Nle⁴, D-Phe⁷]-MSH, and α-MSH. The superpotent analogue of α-MSH ([Nle⁴, D-Phe⁷] -MSH) showed a proliferative and survival enhancing effect on tumor metastasis in vivo with no effect on in vitro tumor cell invasion, with similar effects demonstrated by α-MSH. The effects of these melanotropins on spontaneous metastasis formation appears to be negligible. The A375M and A375P human melanoma cells parallel their metastatic profile in vitro when assayed in MICS. In concert with these studies, the development of a control cell line, comprised of neural crest-derived melanocytes, and the study of their subsequent invasiveness in vitro were pursued. The neural crest-derived melanocytes were unable to invade the basement membranes (BM); although co-culturing neural crest cells with B16F10 melanoma cells produced an effect such that the neural crest cells did significantly invade the BMs. These studies demonstrate the ability of the MICS in vitro invasion assay to discriminate between tumor cells with differing metastatic propensities and could possibly be used in future studies to predict the effectiveness of biological response modifying agents in vivo.