WO2002069998A1

WO2002069998A1 - Regulating vegf expression by a protein of the tis11 family

Info

Publication number: WO2002069998A1
Application number: PCT/EP2001/003432
Authority: WO
Inventors: Jean-Jacques Feige; Sabine Bailly; Anna Chinn; Hervé Prats
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2001-03-06
Filing date: 2001-03-06
Publication date: 2002-09-12

Abstract

The invention relates to the down-regulation of VEGF expression by destabilization of VEGF mRNA with a protein of the TIS11 family. The invention is useful in the treatment of pathologies resulting from VEGF overexpression such as tumor growth, psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, neovascular glaucoma, age-related macular degeneration and rejection of corneal graft.

Description

REGULATING VEGF EXPRESSION BY A PROTEIN OF THE TISll FAMILY

The invention relates to the regulation of angiogenesis, through the control of the expression of vascular endothelial growth factor-A (VEGF-A) . VEGF-A, hereinafter referred to as VEGF, is a heparin-binding glycoprotein that is normally secreted as a homodimer of 34 to 46 kDa by most types of cells, in particular in the pituitary gland, hypothalamus, choroid plexus, renal glomeruli, adrenal cortex, cardiac myocytes, prostate epithelium, semen, endometrium, lung alveolar epithelial cells, and the corpus luteum of the ovaries. It is overexpressed in most human tumors and tumor cell lines, for instance lymphoma, sarcoma, meningioma, glioblastoma, melanoma, breast gastric, kidney, colonic, adrenal, ovarian, lung and thyroid carcinomas, hepatomas and angiosarcomas .

VEGF is an activator of microvascular permeability and a potent mitogen for endothelial cells. It plays an important role in physiological angiogenesis, associated for instance with embryonic development and wound healing, and also in angiogenesis associated with pathological processes such as tumour angiogenesis, diabetic retinopathy, psoriasis, or rheumatoid arthritis.

In particular, VEGF expression plays an important role in promoting tumor angiogenesis and progression of several human malignant tumors.

Targeting the VEGF^' expression and/or function is therefore of major interest for the development of therapies aiming either at promoting or at inhibiting angiogenesis .

It is has been shown that VEGF expression is up- regulated by hypoxia. This up-regulation is at least partially attributed to an increase of transcription and of VEGF mRNA stability. The effect of hypoxia on VEGF transcription appears to be mediated by the binding of 5' flanking elements of the VEGF gene with a hypoxia-regulated transcription factor,- hypoxia-inducible factor 1 or hypoxia- inducible factor 2 (also named EPAS) . It has been reported that the increase of VEGF mRNA stability involved hypoxia- induced proteins interacting with VEGF mRNA. VEGF belongs to a group of genes with labile mRNAs, including cytokines such as interleukin 2 (IL-2), interleukin 3 (I -3), granulocyte-macrophage colony- stimulating-factor (GM-CSF) and tumor necrosis factor alpha (TNF-cc) , and proto-oncogenes , such as c-myc, c~ fos, and c-jun . Many of these mRNAs possess in their 3' untranslated regions (3' UTR) adenylate-uridylate-rich elements (AU-Rich Elements or AREs) comprising the consensus AUUUA motif, which are thought to play a role in the regulation of mRNA stability) .

Many proteins able to bind to the AREs of VEGF mRNA and potentially involved in its stability have been described.

For instance, LEVY et al . (J. Biol . Chem. , 271, 2746-2753, 1996 ) have identified hypoxia-inducible proteins of apparent molecular weights of 17 kDa, 28 kDa, and 32 kDa that bind to the AREs of VEGF mRNA.

CLAFFEY et al . (Mol . Biol. Cell., 9, 469-481,

1998) have identified a 125-bp AU-rich element with a stem loop secondary structure in the 3' untranslated region of the

VEGF mRNA. This element appears to be involved in the stabilization of VEGF mRNA. They also identified several hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa, that bind said AU-rich element. A stabilization of VEGF mRNA has been observed with the protein HuR (LEVY et al . , J. Biol. Chem., 273, 6417- 6423, 1998) and it has been reported that VHL (von Hippel- Lindau) protein promotes rapid degradation of VEGF mRNA (ILIOPOULOS et al . , Proc . Natl . Acad. Sci . USA, 93, 10595- 10599, 1996) .

DIBBENS et al . (Mol. Biol. Cell., 10, 907-919,

1999) report that VEGF mRNA not only contains destabilizing elements in its 3' untranslated region (3 'UTR), but also in its 5 'UTR and coding region. The 1038 nucleotide-long 5 ' -UTR of VEGF has a complex structure, and has been shown to contain two functional internal ribosome entry sites (IRES) (HUEZ et al . , Mol. Cell. Biol., 18, 6178-6190, 1998; STEIN et al . , Mol Cell. Biol, 18, 3112-3119, 1998), involved in the initiation of translation. The identity of the regulatory proteins that bind to these internal ribosome entry sites is still unknown (VAN DER ELDEN and THOMAS, Int. J. Biochem. Cell. Biol., 31, 87-106, 1999). Thus it appears that the regulation of expression of VEGF mRNA is a complex mechanism, resulting from the interaction of many different factors.

In a recent study, the inventors observed that ACTH induces a transient induction of VEGF expression (GAILLARD et al . , J. Cell. Physiol . , 185, 226-234, 2000). The amount of VEGF mRNA reaches a maximum 2h after ACTH treatment, then decreases rapidly.

They have now undertaken the characterization of other ACTHregulated genes, and have observed that ACTH also induces the expression of the zinc finger protein TISllb. This induction starts after 30 min and peaks between 3 and 5h after ACTH addition. On the basis of this observation the inventors have hypothesized that ^' TISllb could act as a regulator of VEGF expression. They have thus found that the protein TISllb destabilizes the VEGF mRNA, and that this destabilization involves sequences of both the 3' and the 5' UTRs .

TISllb, also known as cMG, ERF-1, or Berg-36 belongs to a family of zinc finger proteins containing two tandem fingers of the CCCH type (i.e. having a CX₈CX₅CX₃H motif) . The prototype of this family is TISll (TPA-inducible sequence 11) also known as tristetraprolin (TTP) or Nup475 which was initially identified in fibroblasts as the product of an immediate early response gene induced by insulin, serum or the phorbol ester TPA (VARNUM et al . , Mol. Cell. Biol., 11, 1754-1758, 1991; VARNUM et al . , Oncogene, 4, 119-120, 1989; HERSCHMAN, Annu. Rev. Biochem., 60, 281-319, 1991). TISllb and TISlld (ERF2) are the other two mammalian members of this family. Both are constitutively expressed at a low level, and are inducible by a number of factors (mostly mitogenic) in different cell types (VARNUM et al . , Mol. Cell. Biol., 11, 1754-1758, 1991; BUSTIN et al . , DNA Cell. Biol., 13, 449-459, 1994, GOMPERTS et al . , FEBS Lett., 306, 1-4, 1992) . Alter remaining elusive for several years, the biological function of the TISll family members has begun to be characterized by BLACKSHEAR and colleagues (J. Biol. Chem., 275, 17827-17837, 2000; Science, 281, 1001-1005, 1998; Mol. Cell. Biol., 19, 4311-4323, 1999; Blood, 95, 1891-1899, 2000; J. Biol. Chem., 270, 13341-13347, 1995, Immunity, 4, 445-454, 1966, Mol. Endocrinol . , 10, 140-146, 1996). TISll was shown to be rapidly phosphorylated and translocated from nucleus to cytoplasm in response to mitogens (TAYLOR et al . , J. Biol. Chem., 270, 13341-13347, 1995; TAYLOR et al . , Mol. Endocrinol., 10, 140-146, 1996). More recently, it was reported that TISll, TISllb and TISlld all bind to the AU- rich elements of TNFα, GM-CSF and IL-3 mRNAs in vi tro and induce the destabilization and breakdown of these mRNAs (LAI et al . , J. Biol. Chem., 275, 17827-17837, 2000; CARBALLO et al . , Science, 281, 1001-1005, 1998; LAI et al . , Mol. Cell. Biol., 19, 4311-4323, 1999; CARBALLO et al . , Blood, 95, 1891- 1899, 2000, STOECKLIN et al . , Mol. Cell. Biol., 20, 3753- 3763, 2000) . However, no effect of a member of the TISll family on VEGF mRNA has been reported until now.

The invention relates to the use of a protein of the TISll family for regulating VEGF expression.

More specifically, the invention provides a method for down-regulating VEGF expression by inducing destabilization of VEGF mRNA with a protein of the TISll family. Preferably, said protein is selected among TISll, TISllb and TISlld, and more preferably said protein is TISllb. The present invention also provides a method for treating a pathological condition resulting from VEGF overexpression, wherein said method comprises down-regulating VEGF expression by inducing destabilization of VEGF mRNA with a protein of the TISll family. According to the invention, destabilization of

VEGF mRNA is induced by administration of a protein of the TISll family, to a mammal cell, in particular a human cell, expressing VEGF. Preferably, the administration of said protein of the TISll family is accomplished by introduction into said mammal cell of an expression vector comprising a nucleic acid encoding said protein of the TISll family, operatively linked to a promoter.

According to a preferred embodiment of the invention, said cell is a tumor cell.

Pathological conditions resulting from VEGF overexpression include all diseases involving pathological angiogenesis, in particular tumor growth, psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, neovascular glaucoma, age-related macular degeneration and rejection of corneal graft.

In particular, the invention is useful in antitumoral treatments, for reversing the angiogenic effects of VEGF and thus decreasing in the growth rate of said tumor. The present invention thus provides a method to decrease or suppress the growth of a tumor in a mammal, wherein said method comprises administering to said tumor a composition comprising a protein of the TISll family, wherein said administration results in a destabilization of VEGF mRNA in the cells of said tumor.

A DNA encoding a protein of the TISll family can be introduced and expressed into a cell in vi tro, in vivo or ex vivo in a variety of ways, using conventional molecular biology methods

For instance, general methods for constructing expression vectors, and introducing said vectors into eukaryotic cells are described by SAMBROOK et al . (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring

Harbor Laboratory Press, 1989) .

For _ treating pathological conditions resulting from VEGF overexpression, the DNA encoding the protein of the TISll family will be introduced in a gene therapy vector, that will be used in vivo or ex vivo to transfect target cells wherein a down regulation of expression of VEGF is sought . Gene therapy vectors suitable for use in the method of the invention are known in themselves; they include viral vectors such as adenovirus, adenoassociated virus, and retrovirus, and non-viral vectors such as liposomes, nano- particles, or gold microspheres . Naked DNA comprising a sequence encoding a protein of the TISll family operatively linked to a promoter can also be introduced into the cells by electroporation (VICAT et al . , Human Gene Therapy, 11, 909-

916, 2000) .

Gene therapy vectors and methods more specifically suitable for the administration of anti- angiogenic agents in antitumoral therapy are described for instance by KONG and CRYSTAL (J. Nat. Cane. Inst, 90, 273-

286, 1998) .

The present invention will be further illustrated by the following example and drawings, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of the luciferase reporter gene constructs used to study VEGF mRNA stability. A: pRL-CMV encoding renilla luciferase placed downstream the CMV promoter. B : pRVFV-CMV allowing expression of renilla luciferase under the control of the VEGF 3 ' -UTR, and firefly luciferase under the control of both the VEGF 3 ' - and 5 ' -UTRs . Figure 2 represents luciferase activity in primary cultures of bovine fasciculata cells transfected with 1, 10 or 50 ng of either CMV-TISllb or the control β-galactosidase expression plasmid pRK7βgal, and with either pRL-CMV (control) or pRVFV-CMV (VEGF 3 ' -UTR; VEGF 3' & 5 ' -UTR) . The results are represented as ratios between activities measured in TISllb expressing cells versus β-galactosidase expressing cells.

EXAMPLE: EFFECT OF TIS11B EXPRESSION ON VEGF MRNA STABILITY. Construction of a vector expressing TISllb. TISllb cDNA was obtained by RT-PCR from human

NCI-H295R (ATCC n° CRL-2128) cells. A 262 bp PCR product corresponding to nt 1131-1393 of the human sequence (called ERF-1) was generated by RT-PCR using the primers :

5 ' -ATGGACGTGGGGCTGTCCAG-3 ' and

5 ' -CACGGCATCCACGAGTCC-3 ' with the following program: 94°C for 30 s, 62°C for 45 s, 72°C for 45 s for 35 cycles.

PCR products were cloned into pGEMT-Easy (GENOME EXPRESS, Grenoble, France) and sequenced.

The expression vector CMV-TISllb was obtained by cloning TISllb cDNA obtained by PCR into the pTarget™ vector

(PROMEGA, Charbonnieres, France) , under control of the human cytomegalovirus (CMV) immediate-early enhancer/promoter region.

The vector pRK7lβgal which expresses β-galactosidase under control of CMV immediate-early enhancer/promoter region was used as a control .

Reporter constructs - pRVFV-CMV plasmid is a previously described bicistronic plasmid (HUEZ et al . , Mol. Cell. Biol., 18, 6178- 6190, 1998) that contains two different reporter cistrons, i.e., the renilla and firefly genes coding for luciferase enzyme driven by the CMV immediate -early enhancer/promoter region with the full 5' UTR of VEGF cloned between the two luciferase genes : and the full 3 ' UTR of VEGF cloned behind the firefly luciferase gene. Figure 1 (a) shows a schematic representation of this construct.

This biscistronic vector allows to study the role of the 3 'UTR of VEGF when measuring renilla luciferase activity, and the effect of the 3' and 5' UTR of VEGF when measuring firefly luciferase activity. pRL-CMV plasmid which encodes renilla luciferase under the control of the CMV immediate -early promoter was used as a control. This vector is shown in Figure 1 (b) . Cell transfection:

Bovine adrenocortical (BAC) fasciculata cell primary cultures were prepared as described in (NEGOESCU et al . , Mol. Cell. Endocrinol., 105, 155-163, 1994) and cultured in Ham's F12 medium (Life Technologies, Inc.; Grand Island, NY) supplemented with 12.5% horse serum and 2.5% fetal calf serum.

BAC cells were transfected with the lipocation

Tfx-20™ (PROMEGA, Charbonnieres, France) . Transfections were performed in 24-well plates (10^s cells per well) with 700 ng of pRL-CMV as a control or of pRVFV-CMV and 1, 10, or 50 ng of pRK7βgal or CMV-TISllb.^'

48h after transfection, cells were lysed and luciferase activities were measured with the DUAL-LUCIFERASE^® reporter assay system (PROMEGA, Charbonnieres, France) on a

LUMAT LB 9507 luminometer (EGG BERTHOLD, Bad ildbad,

Germany) .

Results

Results were expressed as RLU of luciferase activity in the presence of CMV-TISllb plasmid over RLU of luciferase activity in the presence of the same amount the pRK7βgal plasmid. Both renilla and firefly luciferase activities are measured sequentially. The results are shown in Figure 2 as the mean ± SD of triplicates from three independent experiments.

These results clearly indicate that TISllb expression induces a dose-dependent decrease of both renilla and firefly luciferase activities. The effect was clearly observable when luciferase mRNA was under the control of the VEGF 3 ' -UTR (30% and 70% inhibition with 10 ng and 50 ng of vector, respectively) and was even more pronounced when luciferase mRNA was under the control of both the VEGF 3 ' - and 5 ' -UTRs (50% and 90% inhibition with 10 ng and 50 ng of vector, respectively) .

Claims

1) A method for down-regulating VEGF expression in a mammal cell, wherein said method comprises inducing destabilization of VEGF mRNA by administration to said cell of a protein of the TISll family.

2) A method of claim 1 wherein the administration of said protein of the TISll family is accomplished by introduction into said mammal cell of an expression vector comprising a nucleic acid encoding said protein of the TISll family, operatively linked to a promoter.

3) A method of claim 1 wherein said cell is a tumor cell .

4) A method for treating a pathological condition resulting from VEGF overexpression in a mammal, wherein said method comprises inducing destabilization of VEGF mRNA in cells of said mammal overexpressing VEGF by administration to said cells of a protein of the TISll family.

5) A method of claim 6 wherein said pathological condition is selected among tumor growth, psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas^', diabetic retinopathy, neovascular glaucoma, age-related macular degeneration and rejection of corneal graft.

6) A method to decrease or suppress the growth of a tumor in a mammal, wherein said method comprises administering to said tumor a composition comprising a protein of the TISll family, wherein said administration results in a destabilization of VEGF mRNA in the cells of said tumor.

7) A method for down-regulating VEGF expression in a mammal cell in vi tro, wherein said method comprises inducing destabilization of VEGF mRNA by administration to said cell of a protein of the TISll family.

8) A method of claim 7 wherein the administration of said protein of the TISll family is accomplished by introduction into said mammal cell of an expression vector comprising a nucleic acid encoding said protein of the TISll family, operatively linked to a promoter. 9) A method of claim 7 wherein said cell is a tumor cell.

10) A method of any of claims 1 to 9, wherein said protein is selected among TlSlla, TISllb and TISlld. 11) Use of a protein of the TISll family for obtaining a therapeutic composition for treating a pathological condition resulting from VEGF overexpression.

12) The use of claim 11 wherein said pathological condition is selected among tumor growth, psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, . neovascular glaucoma, age-related macular degeneration and rejection of corneal graft.

13) The use of any of claims 11 or 12, wherein said protein of the TISll family is selected among TlSlla, TISllb and TISlld.