Patent Application
20070253953
1. Field of Invention
The present invention relates to the identification of genes and their products including their coding protein products that are useful in diagnostics, prognostics and therapeutics of human tumors. In particular, the present invention relates to a set or sets of genes and their products that are associated with tumor suppressor gene PTEN abnormalities, such as PTEN gene deletions, loss of heterozygosity and/or mutations. The present invention relates further to genes and their products that are associated with PTEN-regulated cellular processes, in particular, the PI3K- and/or Akt signal transduction pathways and activations.
2. Description of Related Arts
The PTEN/MMAC1/TEP1 tumor suppressor gene was identified by genomic representational difference analysis (RDA) on human cancer tissues (1), by positional cloning to the genomic locus mutated in multiple advanced cancers (2), and by homologous search for novel protein tyrosine phosphatase (3). This gene is located on human chromosome 10q23, a genomic locus with frequent loss of heterozygosity in multiple advanced cancers. It encodes a protein of 403 amino acids with sequence highly homologous to protein tyrosine phosphatase and the cytoskeletal proteins tensin and auxilin. Germline mutations of PTEN are associated with Cowden and Bannayan-Zonana syndromes, two autosomal dominant disorders characterized as harmartomas with increased susceptibility to cancer (4-6). Somatic mutations of this gene are found in many human cancers, including glioblastoma and prostate cancer with a frequency of up to 50%. Homozygous deletion of the PTEN gene is lethal and heterozygous deletion results in tumor formation in several organs in mice (7-9). These data indicate that PTEN is an important tumor suppressor for a variety of cancers.
One of the well-characterized functions for PTEN is lipid phosphatase activity. This activity dephosphorylates phosphatidylinositol triphosphate at the D3 position, reducing the amount of an important signal transduction molecule produced by PI3K in response to many growth factors, such as insulin-like growth factor 1(IGF-1) that is implicated in tumor formation (10, 11). This PI3K-antagonizing activity in turn inhibits activation of its downstream effector Akt and leads to inhibition of cell survival and proliferation, cellular processes essential for tumor formation and progression (12). In addition to its lipid phosphatase activity, PTEN is also a tyrosine phosphatase that reduces the tyrosine phosphorylation of the focal adhesion kinase (FAK), indicating that PTEN also negatively regulates interactions with the extracellular matrix (13). Furthermore, PTEN deleted mouse fibroblasts have an enhanced cell motility compared to its isogenic wild-type lines. This enhanced cell motility is associated with increased activities of Rac and Cdc42 through its lipid phosphatase activity (14). Taken together, these data indicate that PTEN regulates many cellular processes through a complex map of signal transduction pathways.
Global gene expression analysis is a useful tool to classify and identify tumor types (15, 16), to identify gene involved in tumor formation and progression (17, 18), and predict clinical outcome of cancer patients (19). Recently, it has also been used to identify signatures for tumor metastasis (20). However, its application to identify molecular signatures of a signal transduction pathway involved in tumor formation and progression has not been reported.
The present invention relates to the identification of genes and their products including their coding protein products that are useful in diagnostics, prognostics and therapeutics of human tumors. In particular, the present invention relates to a set or sets of genes and their products that are associated with tumor suppressor gene PTEN abnormalities, such as PTEN gene deletions, loss of heterozygosity and/or mutations. The present invention relates further to genes and their products that are associated with PTEN-regulated cellular processes, in particular, the PI3K- and/or Akt signal transduction pathways and activations.
The present invention utilizes global gene expression profiling analyses employing gene chip technology to identify transcriptional targets downstream of the complex signal transduction pathways of PTEN. Gene expression profiling was performed on prostate cancer and giloblastoma, two cancer types frequently affected by PTEN mutations. These global gene expression analyses identify a molecular signature that can accurately classify tumor samples according to its PTEN status regardless of tumor types. Extensive studies were carried out for IGFBP2 gene and its protein products, the most significant gene in the signature. It was demonstrated that IGFBP2 is biochemically regulated by PTEN and plays a functional role in PTEN function.
In one embodiment of present invention, a set of genes consists of 490 genes as listed in Table 2 with the Gini index number from highest to the lowest were identified and evaluated for their predictive power of associating with the PTEN status in tumors.
In another embodiment of present invention, a set of genes comprising 12 genes with the highest Gini index were identified and evaluated individually and combined for their predictive power of associating with the PTEN status in tumors.
These genes include insulin-like growth factor binding protein 2 or IGFBP2 (Accession numbers X16302 and S37730), a hypothetical protein (Acc# AF052186), TUA8 Cri-du-chat region (Acc# AF009314), dual specificity phosphatase 10 or MPK-5 (Acc# AB026436), Neuralized (Acc# AF029729), regulator of G-protein signaling 1 or RGS-1 (Acc# S59049), expressed in activated T/LAK lymphocytes or LAP-4p (Acc# AB002405), gamma-tubulin complex protein 2 or GCP2 (Acc# AF042379), human AMP deaminase gene or AMPD3 (Acc# U29926), PFTAIRE protein kinase 1 or PFTK1 (Acc# AB020641), and pleckstrin homology, sec 7 and coiled/coid domains 1 or cytohesin 1 (Acc# M85169).
In yet another embodiment of present invention, individual gene of the above said 12 genes is identified as useful in associating with the PTEN status in tumors, thus, the establishment of diagnostic, prognostic and therapeutic values of these genes and/or their RNA transcripts and/or protein products in human tumors associated with PTEN abnormalities.
In yet another embodiment of present invention, the IGFBP2 gene and its RNA and protein products are identified as closely associated with the PTEN gene abnormalities such as deletions, mutations and loss of heterozygosity.
In a further embodiment of present invention, the IGFBP2 gene and its RNA and protein products are identified to be associated with P13K signal transduction pathway, in particular, PI3K activation and inhibition.
In a further embodiment of present invention, the IGFBP2 gene and its RNA and protein products are identified to be associated with Akt signal transduction pathway, in particular, Akt phosphorylation through activation or inhibition.
In one embodiment of present invention, a diagnostic and/or prognostic product comprising an antibody against the IGFBP2 provides diagnostic and/or prognostic value in associating tumor staging and grading in association with PTEN status.
In a further embodiment of present invention, the IGFBP2 gene and its RNA and protein products are useful in the screening and selection of therapeutic useful drugs against human cancers.
In another embodiment of present invention, a diagnostic and/or prognostic product comprising an antibody against the IGFBP2 is useful in screening and selecting a therapeutic drug for treating human cancers.
In further embodiment of present invention, the IGFBP2 gene, its product such as RNA transcript and/or protein product are useful in designing, screening, validating and developing a therapeutically useful drug or means such as a dominant negative IGFBP2 that is useful in abolishing IGFBP2 normal and/or abnormal functions in promoting cancer formation, progression, antisense RNA, antisense oligonucleotide and/or siRNAi compounds, or shRNA gene knockdown technology that suppress or erase IGFBP2 gene expression or reduce its RNA transcript level; antibodies that neutralize IGFBP2 functionalities, gene therapies that embody the IGFBP2 gene.
In another embodiment of present invention, a diagnostic and/or prognostic product comprising an antibody against the IGFBP2 provides diagnostic and/or prognostic value in predicting the effectiveness and/or responsiveness of a therapeutics for treating human cancers.
In one embodiment of present invention, a diagnostic and/or prognostic product comprising a molecular probe in the forms of a nucleic acid molecule such as oligonucleotide, DNA and/or RNA molecule with its nucleotide sequence homologous or complementary to the gene sequence of the IGFBP2 gene provides diagnostic and/or prognostic value in associating tumor staging and grading in association with PTEN status.
In one embodiment of present invention, a diagnostic and/or prognostic product comprising a molecular probe in the forms of a nucleic acid molecule such as oligonucleotide, DNA and/or RNA molecule with its nucleotide sequence homologous or complementary to the gene sequence of the IGFBP2 gene is useful in the screening and selection of therapeutic useful drugs against human cancers.
In one embodiment of present invention, a diagnostic and/or prognostic product comprising a molecular probe in the forms of a nucleic acid molecule such as oligonucleotide, DNA and/or RNA molecule with its nucleotide sequence homologous or complementary to the gene sequence of the IGFBP2 gene provides diagnostic and/or prognostic value in predicting the effectiveness and responsiveness of a therapeutics for human cancers.
In another embodiment of present invention, a diagnostic and/prognostic product comprising a gene probe or an antibody against its product is useful in diagnosis and/or prognosis of human cancers, in selecting and screening a therapeutic compounds of means for treating human cancers and/or in predicting effectiveness and responsiveness of a therapeutic means including a therapeutic drug in the treatment of human cancers. The above said gene is selected from a group of 490 genes enlisted in the Table 2, in particular, is selected from a group of genes consisting the followings 12 genes: insulin-like growth factor binding protein 2 or IGFBP2 (accession numbers X16302 and S37730), a hypothetical protein (ACC# AF052186), TUA8 CRI-DU-CHAT region (ACC#AF009314), dual specificity phosphatase 10 or MPK-5 (ACC# AB026436), neuralized (ACC# AF029729), Regulator of G-Protein Signaling 1 or RGS-1 (ACC#S59049), expressed in activated T/LAK lymphocytes or LAP-4P (ACC# AB002405), gamma-tubulin complex protein 2 or GCP2 (ACC# AF042379), human amp dearninase gene or AMPD3 (ACC# U29926), pftaire protein kinase 1 or PFTK1 (ACC# AB020641), and pleckstrin homology, SEC 7 and coiled/coid domains 1 or cythohesin 1 (ACC# M85169).
The present invention relates with utilizing the above identified genes in Table 2, in particular the above said 12 genes, especially the IGFBP2 gene and its products as drug target in screening and selecting therapeutically useful compounds and/or means for the treatment of human cancers.
To identify transcriptional targets downstream of the complex signal transduction pathways of PTEN, we performed a gene expression profiling on prostate cancer and glioblastoma, two cancer types frequently affected by PTEN mutations. This global gene expression analysis identifies a molecular signature that can accurately classify tumor samples according to its PTEN status regardless of tumor types. We also studied IGFBP2, the most significant gene in the signature. We demonstrated that IGFBP2 is biochemically regulated by PTEN and plays a functional role in PTEN function.
To facilitate understanding of the invention, a number of terms are defined below:
Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is a nucleoside. A nucleoside containing at least one phosphate group bonded to the 3′ or 5′ position of the pentose is a nucleotide.
Base Pair (bp): a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA, uracil (U) is substituted for thymine. Generally the partnership is achieved through hydrogen bonding.
Nucleic Acid: a polymer of nucleotides, either single or double stranded.
Gene: a nucleic acid whose nucleotide sequence codes for an RNA or a polypeptide. A gene can be either RNA or DNA.
cDNA: a single stranded DNA that is homologous to an MrRNA sequence and does not contain any intronic sequences.
Sense: a nucleic acid molecule in the same sequence order and composition as the homolog mRNA. The sense conformation is indicated with a “+”, “s” or “sense” symbol.
Antisense: a nucleic acid molecule complementary to the respective mRNA molecule. The antisense conformation is indicated as a “−” symbol or with a “a” or “antisense” in front of the DNA or RNA, e.g., “aDNA” or “aRNA”.
Template: a nucleic acid molecule being copied by a nucleic acid polymerase. A template can be single-stranded, double-stranded or partially double-stranded, depending on the polymerase. The synthesized copy is complementary to the template, or to at least one strand of a double-stranded or partially double-stranded template. Both RNA and DNA are synthesized in the 5′ to 3′ direction. The two strands of a nucleic acid duplex are always aligned so that the 5′ ends of the two strands are at opposite ends of the duplex (and, by necessity, so then are the 3′ ends).
Nucleic Acid Template: a double-stranded DNA molecule, double stranded RNA molecule, hybrid molecules such as DNA-RNA or RNA-DNA hybrid, or single-stranded DNA or RNA molecule.
Oligonucleotide: a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof
Primer: an oligonucleotide complementary to a template. The primer complexes with the template to yield a primer/template duplex for initiation of synthesis by a DNA polymerase. The primer/template complex is extended during DNA synthesis by the addition of covalently bonded bases linked at the 3′ end, which are complementary to the template. The result is a primer extension product. Virtually all known DNA polymerases (including reverse transcriptases) require complexing of an oligonucleotide to a single-stranded template (“priming”) to initiate DNA synthesis. A primer is selected to be “substantially” or “sufficiently” complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template/primer complex for synthesis of the extension product of the primer.
Complementary or Complementarity or Complementation: used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T” is complementary to the sequence “T-C-A,” and also to “T-C-U.” Complementation can be between two DNA strands, a DNA and an RNA strand, or between two RNA strands. Complementarity may be “partial” or “complete” or “total”. Partial complementarity or complementation occurs when only some of the nucleic acid bases are matched according to the base pairing rules. Complete or total complementarity or complementation occurs when the bases are completely matched between the nucleic acid strands. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as in detection methods that depend on binding between nucleic acids. Percent complementarity or complementation refers to the number of mismatch bases over the total bases in one strand of the nucleic acid. Thus, a 50% complementation means that half of the bases were mismatched and half were matched. Two strands of nucleic acid can be complementary even though the two strands differ in the number of bases. In this situation, the complementation occurs between the portion of the longer strand corresponding to the bases on that strand that pair with the bases on the shorter strand.
Homologous or homology: refers to a polynucleotide sequence having similarities with a gene or mRNA sequence. A nucleic acid sequence may be partially or completely homologous to a particular gene or mRNA sequence, for example. Homology may also be expressed as a percentage determined by the number of similar nucleotides over the total number of nucleotides.
Complementary Bases: nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration.
Complementary Nucleotide Sequence: a sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to that on another single strand to specifically hybridize between the two strands with consequent hydrogen bonding.
Conserved: a nucleotide sequence is conserved with respect to a preselected (reference) sequence if it non-randomly hybridizes to an exact or total complement of the preselected sequence.
Hybridize and Hybridization: the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via complementary base pairing. Where a primer (or splice template) “hybridizes” with target (template), such complexes (or hybrids) are sufficiently stable to serve the priming function required by a DNA polymerase to initiate DNA synthesis. There is a specific, i.e. non-random, interaction between two complementary polynucleotide that can be competitively inhibited.
Nucleotide Analog: a purine or pyrimidine nucleotide that differs structurally from T. G. C, or U, but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
DNA Homolog: a nucleic acid having a preselected conserved nucleotide sequence and a sequence coding for a receptor capable of binding a preselected ligand.
Amplification: nucleic acid replication involving template specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in that they are sought to be sorted out from other nucleic acids. Amplification techniques have been designed primarily for this sorting. Template specificity is achieved in most amplification techniques by the choice of enzyme.
Enzymatic Amplification: a method for increasing the concentration of a segment in a target sequence from a mixture of nucleic acids without cloning or purification.
Polymerase Chain Reaction (PCR): an amplification reaction is typically carried out by cycling i.e., simultaneously performing in one admixture, the first and second primer extension reactions, each cycle comprising polynucleotide synthesis followed by denaturation of the double stranded polynucleotides formed. Methods and systems for amplifying a DNA homolog are described in U.S. Pat. Nos. 4,683,195 and 4,683,202, both to Mullis et al.
Amplifiable Nucleic Acid and Amplified Products: nucleic acids that may be amplified by any amplification method.
DNA-dependent DNA Polymerase: an enzyme that synthesizes a complementary DNA copy from a DNA template. Examples are DNA polymerase 1 from E. coli and bacteriophage T7 DNA polymerase. Under suitable conditions a DNA-dependent DNA polymerase may synthesize a complementary DNA copy from an RNA template.
DNA-dependent RNA Polymerase or Transcriptase: enzymes that synthesize multiple RNA copies from a double stranded or partially double stranded DNA molecule having a promoter sequence. Examples of transcriptases include, but are not limited to, DNA-dependent RNA polymerase from E. coli and bacteriophage T7, T3, and SP6.
RNA-dependent DNA Polymerase or Reverse Transcriptase: enzymes that synthesize a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template. Thus, reverse transcriptases are both RNA-dependent and DNA-dependent DNA polymerases.
RNase H: an enzyme that degrades the RNA portion of an RNA/DNA duplex. RNase H may be an endonuclease or an exonuclease. Most reverse transcriptase enzymes normally contain an RNase H activity. However, other sources of RNase H are available, without an associated polymerase activity. The degradation may result in separation of the RNA from a RNA/DNA complex. Alternatively, the RNase H may simply cut the RNA at various locations such that pieces of the RNA melt off or are susceptible to enzymes that unwind portions of the RNA.
Reverse Transcription: the synthesis of a DNA molecule from an RNA molecule using an enzymatic reaction in vitro. For example, the RNA molecule may be primed with a primer that is complementary to the RNA molecule and the DNA molecule is synthesized by extension using a reverse transcriptase such as the DNA polymerase with reverse transcription activity, MMLV reverse transcriptase, AMV reverse transcriptase, and any other enzyme that has the ability to synthesize a DNA molecule from an RNA molecule template.
In Vitro Transcription: the synthesis of an RNA molecule from a DNA molecule using an enzymatic reaction in vitro. For example, the DNA molecule may be double stranded and comprises an RNA polymerase promoter such as T7, SP6, T3, or any other enzyme. promoter for synthesis of RNA from DNA.
Vector: a recombinant nucleic acid molecule such as recombinant DNA (rDNA) capable of movement and residence in different genetic environments. Generally, another nucleic acid is operatively linked therein. The vector can be capable of autonomous replication in a cell in which case the vector and the attached segment is replicated. One type of preferred vector is an episome, i.e., a nucleic acid molecule capable of extrachromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as “expression vectors”. Particularly important vectors allow cloning of cDNA from mRNAs produced using a reverse transcriptase.
Functional parts: a portion of an intact molecule that retains one or more desired properties of the intact molecules. Thus, for example, an antibody binds an antigen. In that context of the property of binding that antigen, a functional part of an antibody can be any portion of an antibody that binds the cognate antigen. Similarly, a functional part of a nucleic acid that encodes an antibody that binds that antigen is any portion of that nucleic acid that encodes a polypeptide that binds to that antigen.
Antibody: in various grammatical forms as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a combining site for antigen or paratope. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecules, including those portions known in the art as Fab, Fab's (Fab′)2, Fv and scFv.
Immunoreact: in various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.
Cistron: a sequence of nucleotides in a DNA molecule coding for an amino acid residue sequence and including upstream and downstream DNA expression control elements.
Promoter: a nucleic acid to which a polymerase molecule recognizes, perhaps binds to, and initiates synthesis. For the purposes of the instant invention, a promoter can be a known polymerase binding site, an enhancer and the like, any sequence that can initiate synthesis by a desired polymerase.
Knockdown: a method to which a RNA made from a DNA sequence (shRNA) introduced into a cell or a RNA sequence (siRNA) introduced into a cell to initiate degradation of the mRNA of a protein of interest.
The present invention provides a novel method for identifying and selecting genes that associate with PTEN gene abnormalities and/or PTEN-related cellular process, and/or PI3K- and/or Akt-related signal transduction pathway. The present invention combines the following elements as discussed in details thereafter:
1. isolation of nucleic acids (genomic DNAs or mRNAs) from tumor cells, tumor specimens;
2.preparation of tumor samples for probing microarrays or gene chips;
3.performing gene chip hybridization with tumor samples;
4.Random Forest (RF) computational analyses of the gene chip datasets;
5. identifying genes with predictive power for association with PTEN status tumor and cancer cells.
The invention now will be exemplified further in the following non-limiting examples.
We have used microarray technology to identify overexpression of androgen receptor as the general mechanism for hormone refractory prostate cancer. The data indicate that overexpression of androgen receptor is a diagnostic and therapeutic target for hormone refractory prostate cancer and can be used as a screening method for hormone refractory prostate cancer drug development (
To identify transcriptional targets associate with the PTEN tumor suppressor function, we compared the gene expression profiles of 11 tissue samples that have the wild-type PTEN gene to those of 14 samples that have mutated PTEN gene (table 1). These 25 samples include 12 advanced prostate cancer xenografts and 13 glioblastoma tissue samples. The PTEN status of the prostate cancer xenografts were characterized previously (21) and those of the glioblastoma were determined by western blot and genomic DNA sequence analysis. Six of the glioblastoma samples do not express the PTEN protein and, therefore, were defined as PTEN mutant samples. The other seven samples have the wild-type PTEN because they express the PTEN protein and they do not carry point mutation, which was determined by genomic DNA sequence analysis. We used both prostate cancer and glioblastoma tissue samples in order to increase a tissue-independent signature associated with the PTEN status. We adopted a statistic technique called Random Forest (RF) because this method has been designed to analyze data that contain many covariates and relatively few observations (Breiman L, 1999). This technique is ideal to analyze microarray data, in which expression of a large number of genes is observed in a relatively few samples. This approach identified 490 genes that have statistic power in predicting the PTEN status, and ranked each gene according to the significance of its predictive power, which is represented by Gini index (
We next ask how many genes must be included in order to correctly predict the PTEN status, since most of the 490 genes have weak predictive power (their Gini indexes are near zero). We generated an error rate for each gene set by a three-fold cross-validation, in which one-third of the samples were left out as test sets. The first gene in the gene set was the one with the most predictive power (the highest Gini index), and a following gene was added each time according to its ranking of the Gini index. A gene list cannot predict the PTEN status accurately until 12 genes with the highest Gini indexes were included (
Having identified several genes whose expression patterns correlate with the PTEN status, we wish to investigate biochemical regulation and biological role of one of these genes in PTEN function. Because both probe sets in the microarray were identified and both have the highest power in predicting the PTEN status, IGFBP2 was chosen for further study. As the first step, we confirmed the relationship between PTEN mutations and IGFBP2 expression by western blot analysis using whole tissue lysates from prostate cancer xenografts. IGFBP2 protein was detected in PTEN mutated xenografts LAPC9, LUCaP 35, and LNCaP but not in PTEN wild-type tumors LAPC4 and LUCaP23 (
The relationship was extended to other tumors that were not included in the microarray analysis. IGFBP2 was highly expressed in PTEN mutated tumors LAPC3, LAPC12, and LUCaP41, but was not detected in PTEN wild-type tumor LAPC14 (
There is one exception for the association between the PTEN mutations and IGFBP2 expression in glioblastoma samples (
To examine if high level of IGFBP2 is secreted by cells with PTEN mutations, glioblastoma (9L and U251) and prostate cancer cells (LAPC4 and LNCaP) were grown in tissue culture and the IGFBP2 levels were measured by radioimmunoassay. In contrast to less than 20 ng/ml of secreted IGFBP2 by cells with wild-type PTEN genes, the levels of secreted protein in cells with mutated PTEN gene are more than 70 ng/ml (
To examine if serum IGFBP2 levels correlate with PTEN status in tumors, serum levels of human IGFBP2 were measured from mice carrying human prostate and breast cancer xenografts (
To establish a causal role of PTEN loss in IGFBP-2 upregulation, we extended our analysis to an isogenic model. Western blot analysis was performed using the lysates from the PTEN wild-type and deleted isogenic mouse embryonic fibroblasts (MEF) (22). While IGFBP2 protein was barely detectable in PTEN wild-type MEF, PTEN mutant cells produced a high level of IGFBP2 (
To determine if upregulation of the IGFBP2 expression by the PTEN mutations is dependent upon the PI3K/Akt pathway, pharmacological and genetic approaches were employed. When PTEN mutated cells were treated with a pharmacological drug (LY294002) that inhibits the PI3K kinase activity, the production of IGFBP2 was reduced to the basal level (
To determine if IGFBP2 plays a functional role in the PTEN signal transduction pathway, we introduced either PTEN or IGFBP-2 into PTEN null cell lines by lentiviral infection, which gives highly efficient infection rates in prostate cancer epithelial cells (>90%). As reported, re-introduction of the wild-type PTEN decreased growth of PC3 cells by 36% (
To examine if IGFBP2 is involved in the PI3K signaling, a pharmacological approach was employed. Consistent with the result in
To determine how IGFBP2 is involved in Akt function, we made use of gene knockdown technology by shRNA. The shRNA efficiently knockdown IGFBP2 expression, as shown by western blot analysis (
Through random forest and other statistical analysis, we identified upregulation of IGFBP2 expression as the most consistent change associated with PTEN mutations. Among 12559 probe sets in the microarray, both probe sets representing IGFBP2 were identified as the most and the second most significant gene to predict the PTEN status. We demonstrated that IGFBP2 is biochemically regulated by PTEN and PI3K-Akt pathway. Consistent with our finding, it was reported that overexpression of IGFBP2 was only observed in glioblastoma, but not in low- or intermediate-grade gliomas (24). In addition, IGFBP2 overexpression was observed in 50% of glioblastoma. The stage in which IGFBP2 is overexpressed and the percentage of tumors with this gene overexpression coincide with the frequency of PTEN mutations in advanced gliomas (25). Overexpression of IGFBP2 was also identified as the most distinct progression-related expression change in high-grade gliomas in another similar study through cDNA microarrays and tissue arrays (26). This study uncovered that IGFBP2 is a poor prognostic marker for patients with gliomas. While patients with IGFBP2 negative tumors had a mean survival of 75 months, patients with tumors of strong IGFBP2 expression had a mean survival of 23 months. This also coincides with the aggressiveness of PTEN mutated tumors. These data suggest that upregulation of IGFBP2 in PTEN mutated tumors may play an important role in tumor formation and progression. Indeed, forced expression of IGFBP2 partially rescued the inhibitory effect of PTEN and a PI3K inhibitor as well (
We and other have recently demonstrated that tumors with PTEN mutations are more sensitive to drugs such as CCI-779 that targets mTOR, a downstream effector of the PI3K/Akt pathway (22, 27). This effect is later observed in several other studies. These studies suggest that drugs targeting the PI3K/Alkt pathway may only benefit patients who have aberrant PTEN/Akt activities. Since PTEN mutations are carried in less than 50% of tumors even for the most frequently mutated cancer type, the pharmaceutical benefit can be masked by an unselected population. This may explain why CCI-779 and some other drugs targeting the PI3K-Akt pathway fail in clinical trials, even though this drug effectively inhibits PTEN mutated cancer cells. Because IGFBP2 is a serum protein, we envision that the serum level of IGFBP2 can be used to predict PTEN mutations and Akt activation. In support of this notion, we detected high concentrations of human IGFBP2 in condition medium of PTEN mutated cells and also in sera of mice carrying PTEN mutated human tumors. In addition, serum concentration of IGFBP2 was shown to be elevated in 50% of patients with advanced prostate cancer (personal communication, Pinchas Cohen). The stage in which IGFBP2 is overexpressed and the percentage of tumors with this gene overexpression coincide with the frequency of PTEN mutations in advanced prostate cancer in patients. Furthermore, it was reported that patients treated with IGF-l, a stimulus for Akt activation, caused an elevated level of IGFBP2 in serum (28). Serum level of IGFBP2 can also be used to predict if drugs hit targets because overexpression of IGFBP2 can be inhibited by a PI3K inhibitor.
The smallest gene expression signature associated with the PTEN status contained eight down-regulated and four up-regulated genes in PTEN mutated tumors (
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This application is related to U.S. Pat. No. 10/701,490, filed Nov. 5, 2003, the entire contents of which are incorporated herein by reference. Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference herein in their entireties.
cerevisiae VTI1)
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US04/42258 | 12/15/2004 | WO | 7/9/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60530101 | Dec 2003 | US |
