Adenoviral Vectors Expressing Human Endostatin–Angiostatin and Soluble Tie2: Enhanced Suppression of Tumor Growth and Antiangiogenic Effects in a Prostate Tumor Model

Angiogenesis is essential for prostate cancer development and metastasis. Antiangiogenic therapy targeting tumor neovasculature, therefore, represents a promising approach for prostate cancer treatment. We hypothesized that adenoviral-mediated delivery of a combination of antiangiogenic factors might have an enhanced antitumor response. We developed the adenoviral vectors Ad-hEndo-angio, expressing a unique, chimeric human endostatin–angiostatin fusion protein, and Ad-sTie2, expressing a soluble form of endothelium-specific receptor tyrosine kinase Tie2. Matrigel angiogenesis assays using Ad-hEndo-angio revealed significant inhibition of tubular network formation and endothelial sprouting compared to Ad-sTie2. In vivo studies in a bilateral PC-3 tumor xenograft model following either intratumoral or systemic administration of Ad-hEndo-angio led to enhanced tumor growth suppression compared to Ad-sTie2. A novel finding is that an intratumoral, combination therapy employing one-half the dose of Ad-hEndo-angio as well as Ad-sTie2 led to a complete regression of the injected, as well as the contralateral uninjected, tumor and prolonged the tumor-free survival in 80% of the animals. In addition, a novel, real-time, intravital imaging modality was used to monitor antiangiogenic responses following adenoviral-mediated gene transfer. These results suggest that a combinatorial antiangiogenic gene therapy approach involving Ad-hEndo-angio and Ad-sTie2 could become a novel form of treatment for localized human prostate cancer

Alternate processing of Flt1 transcripts is directed by conserved cis-elements within an intronic region of FLT1 that reciprocally regulates splicing and polyadenylation

The vascular endothelial growth factor receptor, Flt1 is a transmembrane receptor co-expressed with an alternate transcript encoding a secreted form, sFlt1, that functions as a competitive inhibitor of Flt1. Despite shared transcription start sites and upstream regulatory elements, sFlt1 is in far greater excess of Flt1 in the human placenta. Phorbol myristic acid and dimethyloxalylglycine differentially stimulate sFlt1 compared to Flt1 expression in vascular endothelial cells and in cytotrophoblasts. An FLT1 minigene construct containing exon 13, 14 and the intervening region, recapitulates mRNA processing when transfected into COS-7, with chimeric intronic sFlt1 transcripts arising by intronic polyadenylation and other Flt1/sFlt1 transcripts by alternate splicing. Inclusion of exon 15 but not 14 had a modest stimulatory effect on the abundance of sFlt1. The intronic region containing the distal poly(A) signal sequences, when transferred to a heterologous minigene construct, inhibited splicing but only when cloned in sense orientation, consistent with the presence of a directional cis-element. Serial deletional and targeted mutational analysis of cis-elements within intron 13 identified intronic poly(A) signal sequences and adjacent cis-elements as the principal determinants of the relative ratio of intronic sFlt1 and spliced Flt1. We conclude that intronic signals reciprocally regulate splicing and polyadenylation and control sFlt1 expression

An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function

Sgk1 is an aldosterone-induced kinase that regulates epithelial sodium channel (ENaC)-mediated Na+ transport in the collecting duct and connecting tubule of the kidney. The NH2 terminus of Sgk1 contains instability motifs that direct the ubiquitination of Sgk1 resulting in a rapidly degraded protein. By bioinformatic analysis, we identified a 5′ variant alternate transcript of human Sgk1 (Sgk1_v2) that is widely expressed, is conserved from rodent to humans, and is predicted to encode an Sgk1 isoform, Sgk1_i2, with a different NH2 terminus. When expressed in HEK293 cells, Sgk1_i2 was more abundant than Sgk1 because of an increased protein half-life and this correlated with reduced ubiquitination of Sgk1_i2 and enhanced surface expression of ENaC. Immunocytochemical studies demonstrated that in contrast to Sgk1, Sgk1_i2 is preferentially targeted to the plasma membrane. When coexpressed with ENaC subunits in FRT epithelia, Sgk1_i2 had a significantly greater effect on amiloride-sensitive Na+ transport compared with Sgk1. Together, the data demonstrate that a conserved NH2-terminal variant of Sgk1 shows improved stability, enhanced membrane association, and greater stimulation of epithelial Na+ transport in a heterologous expression system

An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function

Sgk1 is an aldosterone-induced kinase that regulates epithelial sodium channel (ENaC)-mediated Na+ transport in the collecting duct and connecting tubule of the kidney. The NH2 terminus of Sgk1 contains instability motifs that direct the ubiquitination of Sgk1 resulting in a rapidly degraded protein. By bioinformatic analysis, we identified a 5′ variant alternate transcript of human Sgk1 (Sgk1_v2) that is widely expressed, is conserved from rodent to humans, and is predicted to encode an Sgk1 isoform, Sgk1_i2, with a different NH2 terminus. When expressed in HEK293 cells, Sgk1_i2 was more abundant than Sgk1 because of an increased protein half-life and this correlated with reduced ubiquitination of Sgk1_i2 and enhanced surface expression of ENaC. Immunocytochemical studies demonstrated that in contrast to Sgk1, Sgk1_i2 is preferentially targeted to the plasma membrane. When coexpressed with ENaC subunits in FRT epithelia, Sgk1_i2 had a significantly greater effect on amiloride-sensitive Na+ transport compared with Sgk1. Together, the data demonstrate that a conserved NH2-terminal variant of Sgk1 shows improved stability, enhanced membrane association, and greater stimulation of epithelial Na+ transport in a heterologous expression system

N-Terminal Cleavage and Release of the Ectodomain of Flt1 Is Mediated via ADAM10 and ADAM 17 and Regulated by VEGFR2 and the Flt1 Intracellular Domain

<div><p>Flt is one of the cell surface VEGF receptors which can be cleaved to release an N-terminal extracellular fragment which, like alternately transcribed soluble Flt1 (sFlt1), can antagonize the effects of VEGF. In HUVEC and in HEK293 cells where Flt1 was expressed, metalloprotease inhibitors reduced Flt1 N-terminal cleavage. Overexpression of ADAM10 and ADAM17 increased cleavage while knockdown of ADAM10 and ADAM17 reduced N-terminal cleavage suggesting that these metalloproteases were responsible for Flt1 cleavage. Protein kinase C (PKC) activation increased the abundance and the cleavage of Flt1 but this did not require any residues within the intracellular portion of Flt1. ALLN, a proteasomal inhibitor, increased the abundance of Flt1 which was additive to the effect of PKC. Removal of the entire cytosolic region of Flt1 appeared to stimulate cleavage of Flt1 and Flt1 was no longer sensitive to ALLN suggesting that the cytosolic region contained a degradation domain. Knock down of c-CBL, a ring finger ubiquitin ligase, in HEK293 cells increased the expression of Flt1 although it did not appear to require a previously published tyrosine residue (1333Y) in the C-terminus of Flt1. Increasing VEGFR2 expression increased VEGF-stimulated sFlt1 expression and progressively reduced the cleavage of Flt1 with Flt1 staying bound to VEGFR2 as a heterodimer. Our results imply that secreted sFlt1 and cleaved Flt1 will tend to have local effects as a VEGF antagonist when released from cells expressing VEGFR2 and more distant effects when released from cells lacking VEGFR2.</p></div

Effect of PMA and the C-terminal domain of Flt1 on regulating the abundance and cleavage of Flt1.

<p><b>Panel A and B:</b> HEK293 cells transfected with epitope tagged Flt1 and incubated with PMA (30 nM) and/or GF109 (1 µM) and for 24 hrs. Lysates were immunoblotted with Flag and HA and with HSP90 as a loading control. PMA increases the abundance of full length (FL) Flt1 and increases the cleaved N-terminal fragment in conditioned media (condt. media) and the corresponding C-terminal fragment in HEK293 cell lysates. The effect of PMA is blocked by the PKC inhibitor GF109293X (GF109) indicating that the PMA effect is mediated via activation of PKC. Representative immunoblot in A and quantitative pooled data from 3 experiments are shown in B. *p<0.05, **p<0.001. <b>Panel C and D:</b> HEK293 cells transfected with Flt1 or Flt1 ΔCTD and incubated with PMA and for 24 hrs. PMA has a greater effect on increasing Flt1 ΔCTD abundance and on increasing N-terminal cleavage. Representative immunoblot in C and quantitative pooled data of the N-term fragment in condt. Media from 4 experiments are shown in D. *p<0.05 by Mann Whitney Rank sum test. Small arrowheads in Panel C indicate the position of full length FLT1 (FL FLT1) and of the C-terminal deleted form of Flt1 (Flt1 ΔCTD). <b>Panel E:</b> HEK293 cells transfected with Flt1 ΔCTD and incubated with PMA (30 nM) and/or GM6001 (1 µM) for 24 hrs. Conditioned media (condt. media) was blotted with HA while lysates were blotted with tubulin. PMA increases while GM6001 reduces the abundance the N-terminal HA tagged Flt1 fragment in CM.</p

Effect of the metalloprotease inhibitors, GM6001 and TAPI-1 on Flt1 N-terminal cleavage.

<p><b>Panel A:</b> HUVECs were incubated with GM6001 (10 µg/ml) and PMA (30 nM) for the indicated times. GM6001 significantly reduces the PMA-induced soluble Flt1 levels measured by ELISA in conditioned media (CM). **p<0.001 and *p<0.05, n = 3. <b>Panel B and C:</b> HEK293 cells transiently expressing HA and Flag-tagged Flt1 were treated with metalloproteases inhibitors, GM6001 (10 µg/ml) and TAPI-1 (20 µM) and conditioned media was immunoblotted with HA, the epitope tag at the N-terminal end of Flt1 or with AF321, an antibody that recognizes the N-terninus of Flt1 and/or sFlt1. Both GM6001 and TAPI-1 significantly reduces the abundance of the cleaved N-terminal fragment. Representative immunoblot in B and pooled data quantified by densitometry shown in C. **p<0.001 by Kruskal Wallis ANOVAR, Mean ± SE, n = 3–7.</p

Effect of the metalloproteases, ADAM10 and ADAM17 on Flt1 cleavage in HEK293 cells.

<p><b>Panel A and B:</b> ADAM10 and ADAM17 were co-expressed with Flt1 in HEK293 cells and conditioned media was immunoblotted with HA, the epitope tag at the N-terminal end of Flt1. Lysates were immunoblotted for ADAM 17 and HA labeled ADAM 10 to confirm overexpression and for tubulin as a loading control. Both ADAM10 and ADAM17 significantly increased the abundance of the cleaved N-terminal fragment seen in HEK293 conditioned media (condt. Media). Representative immunoblot in A and pooled data quantified by densitometry shown in B. *p<0.05 by Kruskal Wallis ANOVAR, Mean ± SE, n = 7. <b>Panel C:</b> Efficiency of ADAM10 and ADAM17 mRNA knockdown in HEK293 cells transfected with DsiRNA duplexes against ADAM10 or ADAM17 compared to scrambled negative control duplexes. **p<0.001 by Mann Whitney Rank Sum test, n = 3. <b>Panel D and E:</b> Knockdown of either ADAM10 or ADAM17 significantly reduced the abundance of the cleaved N-terminal Flt1 fragment seen in HEK293 conditioned media (condt media). Immunoblot against ADAM10 and 17 confirms specific KD. Representative immunoblot in D and quantitative pooled data in E are shown. *p<0.05 by Kruskal Wallis ANOVAR, Mean ± SE, n = 7.</p

Effect of c-CBL and the Flt1 Y1333F mutation on Flt1 abundance and cleavage.

<p><b>Panel A:</b> HEK293 cells co-transfected with epitope tagged Flt1 or Flt1 Y1333F and c-CBL GFP wt or GFP and incubated with PMA (30 nM) and/or ALLN (10 µM) and for 24 hrs and then immunoblotted for Flag, HA and tubulin. Neither Flt1 Y1333F nor c-CBL has an effect on the abundance of Flt1 or its response to PMA or ALLN. <b>Panel B, C and D:</b> HEK293 cells co-transfected with epitope tagged Flt1 or Flt1 Y1333F and c-CBL DsiRNA or scrambled DsiRNA and incubated with PMA. Knockdown of c-CBL increases the abundance of full length (FL) Flt1 and Flt1 Y1333F (C) and the N-terminal fragments cleaved from them (D). Representative immunoblot and pooled data from 5 experiments are shown. *p<0.05, **p<0.001 against Flt1+ScrNeg, ^$p<0.05 against Flt + Dsi c-Cbl. <b>Panel E:</b> HEK293 cells co-transfected with epitope tagged Flt1 or Flt1 Y1333F and c-CBL DsiRNA or scrambled DsiRNA and samples were immunoprecipitated with Flag and immunoblotted with c-CBL. C-CBL physically associates with Flt1 and this association is not altered by mutation of a tyrosine at 1333 to phenylalanine.</p