Transforming growth factor beta 1 (TGF-b1) is a potent tumor suppressor but, paradoxically, TGF-b1 enhances tumor growth and metastasis in the late stages of cancer progression. This study investigated the role of TGF-b type I receptor, ALK5, and three mitogen-activated protein kinases (MAPKs) in metastasis by breast cancer cell line MDA-MB-231. We show that autocrine TGF-b signaling in MDA-MB-231 cells is required for tumor cell invasion and tumor angiogenesis. Expression of kinaseinactive ALK5 reduces tumor invasion and formation of new blood vessels within the tumor orthotopic xenografts in severe combined immunodeficiency (SCID) mice. In contrast, constitutively active ALK5-T204D enhances tumor invasion and angiogenesis by stimulating expression of matrix metalloproteinase MMP-9/gelatinase-B. Ablation of MMP-9 in ALK5-T204D cells by RNA interference (RNAi) reduces tumor invasion and tumor growth. Importantly, RNAi-MMP-9 reduces tumor neovasculature and increases tumor cell death. Induction of MMP-9 by TGF-b-ALK5 signaling requires MEK-ERK but not JNK, p38 MAPK or Smad4. Dominant-negative MEK blocks and constitutively active MEK1 enhances MMP-9 expression. However, all three MAPK cascades (ERK, JNK and p38 MAPK) are required for TGF-b-mediated cell migration. Collectively, our results show that TGF-b-ALK5-MAPK signaling in tumor cells promotes tumor angiogenesis and MMP-9 is an important component of this program.

Transforming growth factor-b 1 (TGF-b1) signaling in tumor cells has been implicated in tumor angiogenesis and metastasis by regulating matrix proteolysis. Although MMP-9/gelatinase-B is an important component of these TGF-b1 responses, the mechanism of its regulation is not well understood. Here, we present evidence that TGF-bactivated protein kinase 1 (TAK1) is critical for TGF-b regulation of MMP-9 and the metastatic potential of breast cancer cell line MDA-MB-231. We found that suppression of TAK1 signaling by dominant-negative (dn) TAK1 or RNA interference (siRNA) reduces expression of MMP-9 and tumor cell invasion, without growth inhibition in cell culture. The orthotopic xenograft studies in SCID mice showed that suppression of TAK1 signaling by dn-TAK1 reduces tumor growth and formation of lung metastases. QJ;Dn-TAK1 reduced the proliferation Ki-67 index and neovasculature of orthotopic xenografts. TAK1-mediated regulation of MMP-9 involves NF-jB signaling. Dn-TAK1 reduces NF-jB transcriptional response and inhibition of NF-jB reduces expression of MMP-9 and activity of the MMP-9 promoter reporter. Together, these findings suggest that TAK1 contributes to TGF-b1-mediated tumor angiogenesis and metastasis via a mechanism involving the TAK1-NF-jB-MMP-9 pathway.

BackgroundFolate (vitamin B9) is essential for cellular proliferation as it is involved in the biosynthesis of deoxythymidine monophosphate (dTMP) and s-adenosylmethionine (AdoMet). The link between folate depletion and the genesis and progression of cancers of epithelial origin is of high clinical relevance, but still unclear. We recently demonstrated that sensitivity to low folate availability is affected by the rate of polyamine biosynthesis, which is prominent in prostate cells. We, therefore, hypothesized that prostate cells might be highly susceptible to genetic, epigenetic and phenotypic changes consequent to folate restriction.ResultsWe studied the consequences of long-term, mild folate depletion in a model comprised of three syngenic cell lines derived from the transgenic adenoma of the mouse prostate (TRAMP) model, recapitulating different stages of prostate cancer; benign, transformed and metastatic. High-performance liquid chromatography analysis demonstrated that mild folate depletion (100 nM) sufficed to induce imbalance in both the nucleotide and AdoMet pools in all prostate cell lines. Random oligonucleotide-primed synthesis (ROPS) revealed a significant increase in uracil misincorporation and DNA single strand breaks, while spectral karyotype analysis (SKY) identified five novel chromosomal rearrangements in cells grown with mild folate depletion. Using global approaches, we identified an increase in CpG island and histone methylation upon folate depletion despite unchanged levels of total 5-methylcytosine, indicating a broad effect of folate depletion on epigenetic regulation. These genomic changes coincided with phenotype changes in the prostate cells including increased anchorage-independent growth and reduced sensitivity to folate depletion.ConclusionsThis study demonstrates that prostate cells are highly susceptible to genetic and epigenetic changes consequent to mild folate depletion as compared to cells grown with supraphysiological amounts of folate (2 μM) routinely used in tissue culture. In addition, we elucidate for the first time the contribution of these aspects to consequent phenotype changes in epithelial cells. These results provide a strong rationale for studying the effects of folate manipulation on the prostate in vivo, where cells might be more sensitive to changes in folate status resulting from folate supplementation or antifolate therapeutic approaches.