RESUMO
Vigilin, a nucleocytoplasmic shuttling protein, post-transcriptionally suppresses proto-oncogene c-fms expression (encoding CSF-1R) in breast cancer by binding to a 69 nt cis-acting 3-UTR element in CSF-1R mRNA. CSF-1R is an important mediator of breast cancer development, metastasis, and survival. We confirm that vigilin decreases in vitro reporter luciferase activity as well as the translation rate of target mRNAs. We further explore the mechanism of suppression of CSF-1R. We show that the 69 nt binding element has profound effects on translation efficiency of CSF-1R mRNA, not seen in the presence of mutation of the element. Also, mutation of the 69 nt element in the CSF-1R mRNA 3'UTR both interferes with direct vigilin binding and obviates effect of vigilin overexpression on translational repression of CSF-1R. We show that stable vigilin binding requires the full length 69 nt CSF-1R element, including the 26 nt pyrimidine-rich core. Furthermore, titration of endogenous vigilin and other proteins which bind the 69 nt element, by exogenously introduced CSF-1R mRNA 3'UTR containing the pyrimidine-rich sequence, increases the adhesion, motility, and invasion of breast cancer cells. This phenotypic effect is not seen when the 69 nt element is deleted. Lastly, we are the first to show that human breast tissues exhibit strong vigilin expression in normal breast epithelium. Our pilot data suggest decreased vigilin protein expression, along with shift from the nucleus to the cytoplasmic location, in the transition to ductal carcinoma in situ.
RESUMO
Although glyceraldehyde-3-phosphate dehydrogenase's (GAPDH) predilection for AU-rich elements has long been known, the expected connection between GAPDH and control of mRNA stability has never been made. Recently, we described GAPDH binding the AU-rich terminal 144 nt of the colony-stimulating factor-1 (CSF-1) 3' untranslated region (UTR), which we showed to be an mRNA decay element in ovarian cancer cells. CSF-1 is strongly correlated with the poor prognosis of patients with ovarian cancer. We investigated the functional significance of GAPDH's association with CSF-1 mRNA and found that GAPDH small interfering RNA reduces both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA. CSF-1 mRNA half-lives were decreased by 50% in the presence of GAPDH small interfering RNA. RNA footprinting analysis of the 144 nt CSF-1 sequence revealed that GAPDH associates with a large AU-rich-containing region. The effects of binding of GAPDH protein or ovarian extracts to mutations of the AU-rich regions within the footprint were consistent with this finding. In a tissue array containing 256 ovarian and fallopian tube cancer specimens, we found that GAPDH was regulated in these cancers, with almost 50% of specimens having no GAPDH staining. Furthermore, we found that low GAPDH staining was associated with a low CSF-1 score (P = 0.008). In summary, GAPDH, a multifunctional protein, now adds regulation of mRNA stability to its repertoire. We are the first to evaluate the clinical role of GAPDH protein in cancer. In ovarian cancers, we show that GAPDH expression is regulated, and we now recognize that one of the many functions of GAPDH is to promote mRNA stability of CSF-1, an important cytokine in tumor progression.