CUL5 is required for thalidomide-dependent inhibition of cellular proliferation.

Angiogenesis is essential for cancer metastasis, thus the discovery and characterization of molecules that inhibit this process is important. Thalidomide is a teratogenic drug which is known to inhibit angiogenesis and effectively inhibit cancer metastasis, yet the specific cellular targets for its effect are not well known. We discovered that CUL5 (previously identified as VACM-1), a scaffold protein in E3 ligase complexes, is involved in thalidomide-dependent inhibition of endothelial cell growth. Our results show that in human endothelial cells (HUVEC), thalidomide-dependent decrease in cell growth was associated with decreased nuclear localization of CUL5. In HUVEC transfected with anti-VACM-1 siRNA, thalidomide failed to decrease cell growth. Previously it was established that the antiproliferative effect of CUL5 is inhibited in rat endothelial cells (RAMEC) transfected with mutated CUL5 which is constitutively modified by NEDD8, a ubiquitin-like protein. In this study, the antiproliferative response to thalidomide was compromised in RAMEC expressing mutated CUL5. These results suggest that CUL5 protein is involved in the thalidomide-dependent regulation of cellular proliferation in vitro. Consequently, CUL5 may be an important part of the mechanism for thalidomide-dependent inhibition of cellular proliferation, as well as a novel biomarker for predicting a response to thalidomide for the treatment of disorders such as multiple myeloma and HIV infection.

Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.

VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 (640PKLKRQ646) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence (S730AVACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in S730AVACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and in S730AVACM-1/CUL5 sequences, is critical for their control of cell proliferation.

New insights into the mechanism for VACM-1/cul5 expression in vascular tissue in vivo.

Vasopressin-activated calcium-mobilizing (VACM-1)/cul5 is the least conserved member of a cullin protein family involved in the formation of E3-specific ligase complexes that are responsible for delivering the ubiquitin protein to their target substrate proteins selected for ubiquitin-dependent degradation. This chapter summarizes work to date that has focused on VACM-1/cul5's tissue-specific expression in vivo and on its potential role in the control of specific cellular signaling pathways in those structures. As mammalian cells may contain hundreds of E3 ligases, identification VACM-1/cul5 as a specific subunit of the system that is expressed in the endothelium and in collecting tubules, structures known for their control of cellular permeability, may have significant implications when designing studies to elucidate the mechanism of water conservation. For example, VACM-1/cul5 expression is affected by water deprivation in some tissues and there is a potential relationship between neddylated VACM-1/cul5 and aquaporins.

Aquaporin-2 levels in vitro and in vivo are regulated by VACM-1, a cul 5 gene.

BACKGROUND: In the renal collecting duct, vasopressin regulates water permeability by a process that involves stimulation of adenylyl cyclase activity, cAMP production and subsequent translocation of water channel aquaporin-2 (AQP2) into the apical plasma membrane. We have previously shown that in cos 1 cells in vitro, both adenylyl cyclase activity and cAMP production can be regulated by VACM-1, a cul 5 gene that forms complexes involved in protein ubiquitination and subsequent degradation. METHODS: To extend these observations further, the effects of changes in hydration state on the expression of VACM-1 at the mRNA and the protein level were examined in rats deprived of water (WD) for 24 hrs. RESULTS: In the kidney of WD rats Western blot analyses of kidney tissue showed that the decrease in VACM-1 protein concentration was correlated with the increase in the AQP2 protein level. The immunostaining data suggested that VACM-1/cul5 may be decreased in renal collecting duct but increases in the vasculature of the inner medullary region in response to WD. To determine the possible consequences of the WD dependent decrease in VACM-1/cul5, we next examined the effects of VACM-1 expression on AQP2 protein in vitro. Immunocytochemistry and Western blot analyses data indicate that VACM-1/cul5 expression in MDCK line stably expressing AQP2 gene and in cos 1 cells co-transfected with the AQP2 and VACM-1/cul5 cDNAs decreased AQP2 protein concentration when compared to the vector transfected control groups. CONCLUSION: In summary, our data demonstrate that VACM-1 is involved in the regulation of AQP2 protein concentration and may play a role in regulating water balance.

VACM-1/cul5 expression in vascular tissue in vivo is induced by water deprivation and its expression in vitro regulates aquaporin-1 concentrations.

VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24 h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24 h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.

Mutational analysis of VACM-1/cul5 exons in cancer cell lines.

VACM-1, a cul-5 gene product, functions via an E3 ligase complex and when overexpressed, has an antiproliferative effect in many cell types. Overexpression of VACM-/cul5 cDNA mutated at the PKA-specific phosphorylation site at Ser730 reversed this phenotype. These effects are associated with the appearance of larger M(r) species subsequently identified as a Nedd8-modified VACM-1/cul5. Although decreased levels of VACM-1 mRNA detected in several cancers and cancer cell lines may explain the progression of cell growth, possible genetic and epigenetic changes in its sequence have not been analyzed. We hypothesized that in rapidly proliferating cells, VACM-1/cul5 may be mutated at either the PKA-specific phosphorylation site or the consensus neddylation site. We used RT-PCR and PCR, to amplify and to sequence mRNA and genomic DNA, respectively. To date we have sequenced all 19 coding exons of the VACM-1/cul5 gene in T47D breast cancer cells, U138MG glioma cells, ACHN renal cancer cells, and OVCAR-3 ovarian cancer cells. Our results indicate that in those cells VACM-1/cul5 is not mutated at the putative phosphorylation or the neddylation site. We have found one silent mutation in the genomic DNA isolated from U138MG, ACHN, and OVCAR-3 cell lines, but not from T47D cells. Our work suggests that in T47D breast cancer cells biologic activity of VACM-1/cul5 may be regulated by posttranslational modifications.

Resveratrol enhances anti-proliferative effect of VACM-1/cul5 in T47D cancer cells.

Vasopressin-activated calcium-mobilizing (VACM-1) protein is a cul-5 gene product that forms complexes with a subclass of ubiquitin E3 ligases involved in proteasomal protein degradation. The expression of VACM-1 cDNA in the T47D breast cancer cell line inhibits growth and decreases phosphorylation of mitogen activated protein kinase. Factors that regulate expression or stability of VACM-1 protein have not been identified, however. In our search to identify drugs/substances that may control VACM-1 protein expression, we examined the effects of resveratrol (trans-3,5,4'-trihydroxystilbene), a natural component in the human diet which inhibits tumor initiation and promotion. CMV vector and VACM-1 cDNA stably transfected T47D breast cancer-derived cells were treated with resveratrol and cell growth and VACM-1 protein concentrations were measured. Since the cellular mechanism of resveratrol-dependent inhibition of cell growth also involves the regulation of estrogen receptors, the effect of 17-ß-estradiol and resveratrol on ERα levels and on cell growth was examined in control and in VACM-1 cDNA transfected cells. Our results demonstrate that antiproliferative effect of resveratrol observed in the control T47D cancer cells was significantly enhanced in VACM-1 cDNA transfected T47D cells. Western blot results indicated that resveratrol increased VACM-1 protein concentration. Finally, treatment with resveratrol for 24 and 48 h attenuated 17-ß-estradiol induced increase in cell growth both in control and in VACM-1 cDNA transfected cells. The effect was significantly higher in the VACM-1 cDNA transfected cells when compared to controls. These results indicate that the antiproliferative effect of resveratrol may involve induction of VACM-1/cul5.

Estrogen-dependent growth and estrogen receptor (ER)-alpha concentration in T47D breast cancer cells are inhibited by VACM-1, a cul 5 gene.

Vasopressin-activated calcium mobilizing receptor (VACM-1)/cullin 5 (cul 5) inhibits growth when expressed in T47D breast cancer cells by a mechanism that involves a decrease in MAPK phosphorylation and a decrease in the early growth response element (egr-1) concentration in the nucleus. Since both MAPK and egr-1 pathways can be regulated by 17beta-estradiol, we next examined the effects of VACM-1 cDNA expression on estrogen-dependent growth in T47D cells and on estrogen receptor (ER) concentrations. Our results demonstrate that in T47D cells, both basal and 17beta-estradiol-dependent increase in cell growth and MAPK phosphorylation were inhibited in cells transfected with VACM-1 cDNA. Further, Western blot and immunocytochemistry data analyses indicate that ER concentrations and its nuclear localization are significantly lower in cells transfected with VACM-1 cDNA when compared to controls. These data indicate that in the T47D cancer cell line VACM-1 inhibits growth by attenuating estrogen-dependent signaling responses. These findings may have implications in the development of cancer treatments.

Truncated form of VACM-1/cul-5 with an extended 3' untranslated region stimulates cell growth via a MAPK-dependent pathway.

We have sequenced a 4.9kb clone (KLB22) which shares 99% sequence homology with the rabbit vasopressin-activated calcium mobilizing (VACM-1) protein. The 5' terminus sequence of KLB22 cDNA (nucleotides 1-1961) is continuous and overlapping with nucleotides 1226-3186 of the VACM-1 cDNA sequence. The 3'UTR of KLB22 cDNA extends beyond the 3'UTR of VACM-1 by 2999nt. KLB22 cDNA encodes a 497 amino acid protein, which putatively begins at Met 284 of the 780 amino acid VACM-1 protein. The in vitro translation of KLB22 cDNA yields a 59kDa protein. When expressed in cos-1 cells, the truncated VACM-1 protein localizes to the nucleus. KLB22 cDNA transfected cells show increased growth rates and increased levels of phosphorylated MAPK when compared to the vector or to VACM-1 cDNA transfected cells. Finally, in vivo, KLB22 protein expression is tissue specific and can be detected in kidney and in heart atrium. These results suggest that truncated VACM-1 cDNA (KLB22) increases cell proliferation through a MAPK pathway.

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene.

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6-6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.