SUMMARY Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors, p35 and p25, are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.

Semaphorin 3B (SEMA3B) is a secreted member of the semaphorin family, important in axonal guidance. We and others have shown that SEMA3B can act as a tumor suppressor by inducing apoptosis either by reexpression in tumor cells or applied as a soluble ligand. The common method of inactivation of SEMA3B is by allele loss and tumor-acquired promoter methylation. We studied the mechanism of SEMA3B-induced tumor cell apoptosis and found that vascular endothelial growth factor (VEGF) 165 significantly decreased the proapoptotic and antimitotic effect of transfected or secreted SEMA3B on lung and breast cancer cells. VEGF 165 binds to neuropilin, receptors for SEMA3B, and we found that SEMA3B competed for binding of 125 I-VEGF165 to lung and breast cancer cells. We also found that small interfering RNA knockdown of tumor-produced VEGF-A or the use of an anti-VEGF neutralizing antibody (Ab) significantly inhibited tumor cell growth in vitro. By contrast, VEGF 121, a VEGF variant that lacks binding to neuropilin (NP)-1 or NP-2 receptors, was not expressed in tumor cells and had no effect on SEMA3B growth-suppressing activities. In conclusion, we hypothesize that VEGF 165, produced by tumor cells, acts as an autocrine survival factor and that SEMA3B mediates its tumor-suppressing effects, at least in part, by blocking this VEGF autocrine activity.

Cyclin D1 gene expression is induced by 17␤-estradiol (E2) in human breast cancer cells and is important for progression of cells through the G 1 phase of the cell cycle. The mechanism of activation of cyclin D1 is mitogen-and cell context-dependent, and this study describes the role of multiple promoter elements required for induction of cyclin D1 by E2 in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Transcriptional activation of cyclin D1 by E2 was dependent, in part, on a proximal cAMP-response element at ؊66, and this was linked to induction of protein kinase A-dependent pathways. These results contrasted to a recent report showing that induction of cyclin D1 by E2 in ER-positive MCF-7 and HeLa cells was due to up-regulation of c-jun and subsequent interaction of c-Jun-ATF-2 with the CRE. Moreover, further examination of the proximal region of the cyclin D1 promoter showed that three GCrich Sp1-binding sites at ؊143 to ؊110 were also E2-responsive, and interaction of ER␣ and Sp1 proteins at these sites was confirmed by electromobility shift and chromatin immunoprecipitation assays. Thus, induction of cyclin D1 by E2 in ZR-75 cells is regulated through nuclear ER␣/Sp1 and epigenetic protein kinase A activation pathways, and our results suggest that this mechanism may be cell context-dependent even among ER-positive breast cancer cell lines.Mitogen stimulation of cell growth is accompanied by the coordinate expression of multiple genes and pathways including those required for different phases of cell cycle progression (1-7). Cyclin D1 is induced early in the G 1 phase of the cell cycle, and cyclin D1-cyclin-dependent kinase complexes are important for phosphorylation of several key substrates involved in cell proliferation including retinoblastoma protein and other pocket proteins. The critical role for cyclin D1 in the rate of progression of cells through G 1 has stimulated studies on factors that regulate cyclin D1 gene expression in various cell types. Transcriptional activation of cyclin D1 depends in part on interaction of trans-acting factors with elements in the cyclin D1 gene promoter; it is clear from promoter analysis studies that the assembly of transcription factors is highly variable and dependent on multiple factors including the mitogen and cell context (8 -20). For example, p21 ras and p300 expression activated constructs containing cyclin D1 gene promoter inserts in JEG-3 human trophoblasts through interactions of proteins at a distal AP-1-like sequence at Ϫ954 in the promoter (13). Overexpression of p60 v-src in MCF-7 breast cancer cells also activates cyclin D1 and involves activation of a cAMP-response element-binding protein (CREB) 1 and activating transcription factor-2 (ATF-2) which interacts with a CRE at Ϫ66 in the cyclin D1 promoter (16).Cyclin D1 protein is overexpressed in ϳ50% of mammary carcinomas (21-23), and 17␤-estradiol (E2) induces cyclin D1 gene expression in estrogen receptor (ER)-positive human breast cancer cell lines (24 -29). Cyclin D1 also directly binds...

Because metastasis contributes significantly to cancer mortality, understanding its mechanisms is crucial to developing effective therapy. Metastasis is facilitated by lymphangiogenesis, the growth of new intratumoral or peritumoral lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor A (VEGF-A) is a well-known angiogenic factor. Increasing evidence implicates VEGF-A in lymphangiogenesis, although the mechanism of its pro-lymphangiogenic effect is poorly understood. We examined the effect of the anti-VEGF-A neutralizing antibody 2C3 on tumor lymphangiogenesis and metastasis in an orthotopic breast carcinoma model using MDA-MB-231 cells and its luciferase-tagged derivative, 231-Luc 1 cells. Anti-VEGF-A antibody therapy reduced blood and lymphatic vessel densities by 70% and 80%, respectively, compared with the control antibody. Treatment with 2C3 antibody also decreased incidence of lymphatic and pulmonary metastases by 3.2-and 4.5-fold, respectively. Macrophage infiltration was reduced in 2C3-treated tumors by 32%, but VEGF-C expression was unchanged. In contrast, neoplastic cells and blood vessels in tumors from 2C3-treated mice expressed significantly less angiopoietin-2 (Ang-2) than tumors from control mice. The reduction in Ang-2 was associated with inhibition of VEGFR-3 expression in intratumoral lymphatic endothelial cells. Both VEGF-A and Ang-2 upregulated the expression of VEGFR-3 in cultured lymphatic endothelial cells. VEGF-A induced proliferation of lymphatic endothelial cells was reduced by 50% by soluble Tie-2, suggesting that Ang-2 is an intermediary of the pro-lymphangiogenic VEGF-A effect. These results suggest a novel mechanism by which anti-VEGF-A therapy may suppress tumor lymphangiogenesis and subsequent metastasis supporting the use of anti-VEGF-A therapy to control metastasis clinically.

Semaphorin 3B (SEMA3B), located at 3p21.3, is a secreted member of the semaphorin family important in axonal guidance. SEMA3B undergoes allele and expression loss in lung and breast cancer and can function as a tumor suppressor. Previously, we found that SEMA3B induces apoptosis in tumor cells either by reexpression or when applied as a soluble ligand. SEMA3B-induced apoptosis was mediated, in part, by blocking vascular endothelial growth factor autocrine activity in tumor cells. In the current study, treatment of lung and breast cancer cells with picomolar concentrations of soluble SEMA3B inhibited their growth; induced apoptosis; and was associated with decreased Akt phosphorylation, increase in cytochrome c release and caspase-3 cleavage, as well as increased phosphorylation of several proapoptotic proteins, including glycogen synthase kinase-3B, FKHR, and MDM-2. Lung and breast cancer lines resistant to SEMA3B did not show these signaling changes and a tumor-derived missense SEMA3B mutant was inactive in this regard, providing specificity. SEMA3B-mediated inhibition of proliferation and induction of apoptosis in cancer cells were blocked by expressing a constitutively active Akt mutant and are linked to tumor cell expression of neuropilin-1 (Np-1). SEMA3B-insensitive Np-1-negative tumor cells acquired sensitivity to SEMA3B after forced expression of Np-1, whereas SEMA3B-sensitive Np-1-positive tumor cells lost sensitivity to SEMA3B after knockdown of Np-1 by small interfering RNA. We conclude that SEMA3B is a potential tumor suppressor that induces apoptosis in SEMA3B-inactivated tumor cells through the Np-1 receptor by inactivating the Akt signaling pathway. [Cancer Res 2008;68(20):8295-303]