Ho Hyung Woo

Regulation of colony stimulating factor-1 expression and ovarian cancer cell behavior in vitro by miR-128 and miR-152

BackgroundColony stimulating factor-1 (CSF-1) plays an important role in ovarian cancer biology and as a prognostic factor in ovarian cancer. Elevated levels of CSF-1 promote progression of ovarian cancer, by binding to CSF-1R (the tyrosine kinase receptor encoded by c-fms proto-oncogene).Post-transcriptional regulation of CSF-1 mRNA by its 3’ untranslated region (3’UTR) has been studied previously. Several cis-acting elements in 3’UTR are involved in post-transcriptional regulation of CSF-1 mRNA. These include conserved protein-binding motifs as well as miRNA targets. miRNAs are 21-23nt single strand RNA which bind the complementary sequences in mRNAs, suppressing translation and enhancing mRNA degradation.ResultsIn this report, we investigate the effect of miRNAs on post-transcriptional regulation of CSF-1 mRNA in human ovarian cancer. Bioinformatics analysis predicts at least 14 miRNAs targeting CSF-1 mRNA 3’UTR. By mutations in putative miRNA targets in CSF-1 mRNA 3’UTR, we identified a common target for both miR-128 and miR-152. We have also found that both miR-128 and miR-152 down-regulate CSF-1 mRNA and protein expression in ovarian cancer cells leading to decreased cell motility and adhesion in vitro, two major aspects of the metastatic potential of cancer cells.ConclusionThe major CSF-1 mRNA 3’UTR contains a common miRNA target which is involved in post-transcriptional regulation of CSF-1. Our results provide the evidence for a mechanism by which miR-128 and miR-152 down-regulate CSF-1, an important regulator of ovarian cancer.

Posttranscriptional suppression of proto-oncogene c-fms expression by vigilin in breast cancer

ABSTRACT cis-acting elements found in 3′-untranslated regions (UTRs) are regulatory signals determining mRNA stability and translational efficiency. By binding a novel non-AU-rich 69-nucleotide (nt) c-fms 3′ UTR sequence, we previously identified HuR as a promoter of c-fms proto-oncogene mRNA. We now identify the 69-nt c-fms mRNA 3′ UTR sequence as a cellular vigilin target through which vigilin inhibits the expression of c-fms mRNA and protein. Altering association of either vigilin or HuR with c-fms mRNA in vivo reciprocally affected mRNA association with the other protein. Mechanistic studies show that vigilin decreased c-fms mRNA stability. Furthermore, vigilin inhibited c-fms translation. Vigilin suppresses while HuR encourages cellular motility and invasion of breast cancer cells. In summary, we identified a competition for binding the 69-nt sequence, through which vigilin and HuR exert opposing effects on c-fms expression, suggesting a role for vigilin in suppression of breast cancer progression.

Regulation of non-AU-rich element containing c-fms proto-oncogene expression by HuR in breast cancer

The role of RNA-binding proteins in cancer biology is recognized increasingly. The nucleocytoplasmic shuttling and AU-rich RNA-binding protein HuR stabilizes several cancer-related target mRNAs. The proto-oncogene c-fms, whose 3′untranslated region (3′UTR) is not AU-rich, is associated with poor prognosis in breast cancer. Using a large breast-cancer tissue array (N=670), we found nuclear HuR expression to be associated with nodal metastasis and independently with poor survival (P=0.03, RR 1.45), as well as to be co-expressed with c-fms in the breast tumors (P=0.0007). We described c-fms mRNA as a direct target of HuR in vivo, and that HuR bound specifically to a 69-nt region containing ‘CUU’ motifs in 3′UTR c-fms RNA. Overexpressing or silencing HuR significantly up- or down-regulated c-fms RNA expression, respectively. We also found that known glucocorticoid stimulation of c-fms RNA and protein is largely dependent on the presence of HuR. HuR, by binding to the 69-nt wild type, but not mutant, c-fms sequence can regulate reporter gene expression post-transcriptionally. We are the first to describe that HuR can regulate gene expression by binding non-AU-rich sequences in 3′UTR c-fms RNA. Collectively, our findings suggest that HuR plays a supportive role for c-fms in breast cancer progression by binding a 69-nt element in its 3′UTR, thus regulating its expression.

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of nucleolins actions on CSF-1 mRNA and describe the dependence of miR-130a- and miR-301a-directed CSF-1 mRNA decay and inhibition of ovarian cancer cell motility on nucleolin.

Inhibition of the c-fms proto-oncogene autocrine loop and tumor phenotype in glucocorticoid stimulated human breast carcinoma cells.

The c-fms proto-oncogene encoded CSF-1 receptor and its ligand represent a feedback loop, which in a paracrine manner, is well known to promote spread of breast cancers. The role of the autocrine feedback loop in promotion of breast tumor behavior, in particular in vitro, is less well understood. The physiologic stimulation of c-fms expression by glucocorticoids (GCs) in vitro and in vivo magnifies the tumor promoting effect seen in these cells from activated c-fms signaling by CSF-1. Targeted molecular therapy against c-fms could therefore abrogate both complementary feedback loops. Using breast cancer cells endogenously co-expressing receptor and ligand, we used complementary approaches to inhibit c-fms expression and function within this autocrine pathway in the context of GC stimulation. Silencing RNA (shRNA), antisense oligonucleotide therapy (AON), and inhibition of c-fms signaling, were all used to quantitate inhibition of GC-stimulated adhesion, motility, and invasion of human breast cancer cells in vitro. shRNA to c-fms downregulated GC-stimulated c-fms mRNA by fourfold over controls, correlating with over twofold reduction in cellular invasiveness. AON therapy was also able to inhibit GC stimulation of c-fms mRNA, and resulted in threefold less invasiveness and 1.5 to 2-fold reductions in adhesion and motility. Finally, the small-molecule c-fms inhibitor Ki20227 was able to decrease in a dose–response manner, breast cancer cell invasion by up to fourfold. Inhibition of this receptor/ligand pair may have clinical utility in inhibition of the autocrine as well as the known paracrine interactions in breast cancer, thus further supporting use of targeted therapies in this disease.

Autocrine inhibition of the c-fms proto-oncogene reduces breast cancer bone metastasis assessed with in vivo dual-modality imaging

Breast cancer cells preferentially home to the bone microenvironment, which provides a unique niche with a network of multiple bidirectional communications between host and tumor, promoting survival and growth of bone metastases. In the bone microenvironment, the c-fms proto-oncogene that encodes for the CSF-1 receptor, along with CSF-1, serves as one critical cytokine/receptor pair, functioning in paracrine and autocrine fashion. Previous studies concentrated on the effect of inhibition of host (mouse) c-fms on bone metastasis, with resulting decrease in osteolysis and bone metastases as a paracrine effect. In this report, we assessed the role of c-fms inhibition within the tumor cells (autocrine effect) in the early establishment of breast cancer cells in bone and the effects of this early c-fms inhibition on subsequent bone metastases and destruction. This study exploited a multidisciplinary approach by employing two non-invasive, in vivo imaging methods to assess the progression of bone metastases and bone destruction, in addition to ex vivo analyses using RT-PCR and histopathology. Using a mouse model of bone homing human breast cancer cells, we showed that an early one-time application of anti-human c-fms antibody delayed growth of bone metastases and bone destruction for at least 31 days as quantitatively measured by bioluminescence imaging and computed tomography, compared to controls. Thus, neutralizing human c-fms in the breast cancer cell alone decreases extent of subsequent bone metastasis formation and osteolysis. Furthermore, we are the first to show that anti-c-fms antibodies can impact early establishment of breast cancer cells in bone.

Phenotype of vigilin expressing breast cancer cells binding to the 69 nt 3′UTR element in CSF-1R mRNA

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.

Post-Transcriptional Regulation of Proto-Oncogene c-fms in Breast Cancer

In the development and progression of breast cancers, both the c-fms proto-oncogene (which encodes the tyrosine kinase receptor for CSF-1) as well as CSF-1 (colony stimulating factor1), play an important role. Evidence from transgenic models suggests that c-fms encodes for the sole receptor for CSF-1 (Dai et al, 2002). We and others have found that c-fms and/or CSF1 are expressed by the tumor epithelium in several human epithelial cancers (Kacinski et al, 1988, 1990, 1991; Rettenmier et al, 1989; Filderman et al, 1992; Ide et al, 2002); elevated levels of c-fms and CSF-1 are associated with poor prognosis (Kacinski et al, 1988; Tang et al, 1990; Price et al, 1993; Chambers et al, 1997, 2009; Scholl et al, 1993; Kluger et al, 2004; Sapi 2004). In human breast cancer, 94% of in situ and invasive lesions express c-fms (Kacinski et al, 1991; Flick et al, 1997), while 36% express both CSF-1 and c-fms (Kacinski et al, 1991; Scholl et al, 1993). Among breast cancer patients, serum levels of CSF-1 are frequently elevated in those with metastases (Kacinski et al, 1991). In breast tumors, nuclear CSF-1 staining is associated with poor survival (Scholl et al, 1994), and c-fms expression confers an increased risk for local relapse (Maher et al, 1998). In a large breast cancer tissue array, c-fms (Kluger et al, 2004) is strongly associated with lymph node metastasis, and poor survival. This strong correlation with prognosis suggests an etiologic role for c-fms/CSF-1 in tumor invasion and metastasis.