Patent Application
20140030716
The present invention relates to a novel tumor marker, DEP domain containing 6 (DEPDC6) protein, and its monoclonal antibodies. The present invention also relates to the use of DEPDC6 protein and its monoclonal antibodies for the identification and/or treatment of cancer, especially to the diagnostic of liver cancer.
Glycine N-methyltransferase (GNMT), also known as a 4S polycyclic aromatic hydrocarbon (PAH) binding protein, has multiple functions. In addition to acting as a major folate binding protein (Yeo E J, et al. Proc Natl Acad Sci USA 1994; 91:210-214), it also regulates the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) by catalyzing sarcosine synthesized from glycine (Kerr S J. J Biol Chem 1972; 247:4248-4252). We previously reported that the GNMT is down-regulated in HCC (Liu H H, et al. J Biomed Sci 2003; 10:87-97). Results from a genetic epidemiological study indicate that GNMT is a tumor susceptibility gene for liver cancer (Tseng T L, et al. Cancer Res 2003; 63:647-654). In addition, we reported that GNMT binds benzo(a)pyrene and prevents DNA-adduct formation (Chen S Y, et al. Cancer Res 2004; 64:3617-3623).
We recently reported that GNMT overexpression results in shifts in aflatoxin B1 (AFB1) detoxification pathways, reduced AFB1-DNA adduct formation, and protection against liver carcinogenesis in vivo. We have also reported that both male and female Gnmt −/− mice portray chronic hepatitis and HCC with very high penetrance. However, how GNMT played in liver hepatocarcinogensis remains unclear. To address this issue, we used full length human GNMT as bait in a yeast two-hybrid screen system with a human kidney cDNA library and identified DEP domain containing 6 (DEPDC6) as a GNMT binding proteins.
Peterson et al. recently reported that DEPTOR (DEPDC6) is frequently overexpressed in multiple myelomas, but is low in most cancers including bladder, cervical, prostate, head and beck, thyroid, skin and brain cancer. They identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt (see, Peterson T R, et al. Cell. 2009; 137:873-886). However, in that report, the expression results of different organs other than myeloma were collected form microarray database. Also they did not detect the protein expression level of DEPDC6 in other tissues.
mTOR is a highly conserved protein kinase located downstream of PI3K and Akt. Its activation is associated with cell growth, survival, and proliferation. mTOR is found in two functionally and structurally distinct complexes, labeled mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The dysregulation of mTORC 1 signaling is frequently observed in human cancer tissues. The Wnt signaling pathway plays an important role in embryonic development, regulating cell proliferation and cellular polarity and determining cell fate (Logan, C. and Nusse, R. Annu Rev Cell Dev Biol 2004; 20:781-810). When the Wnt ligand binds to Frizzled receptor its signals are transduced to DVL, followed by the inactivation of β-catenin degradation complexes. The stabilization of β-catenin supports its association with T-cell factor (Tcf) in cell nuclei and activates targeted genes.
The present invention first identified DEP domain containing 6 (DEPDC6) as a GNMT binding proteins, and examined the expression level of DEPDC6 in tumorous tissues from liver and other organs. The present invention provides the use of DEPDC6 in diagnostic of cancerous disease, especially liver cancer.
In one aspect, the present invention features a novel tumor marker, DEP domain containing 6 (DEPDC6) protein, which is a GNMT binding protein and comprises the amino acid sequence described in SEQ ID NO: 1 (MEEGGSTGSAGSDSSTSGSGGAQQRELERMAEVLVTGEQLRLRLHEEK VIKDRRHHLKTYPNCFVAKELIDWLIEHKEASDRETAIKLMQKLADRGII HHVCDEHKEFKDVKLFYRFRKDDGTFPLDNEVKAFMRGQRLYEKLMSP ENTLLQPREEEGVKYERTFMASEFLDWLVQEGEATTRKEAEQLCHRLM EHGIIQHVSSKHPFVDSNLLYQFRMNFRRRRRLMELLNEKSPSSQETHDS PFCLGKQSHDNRKSTSFMSVSPSKEIKIVSAVRRSSMSSCGSSGYFSSSPT LSSSPPVLCNPKSVLKRPVTSEELLTPGAPYARKTFTIVGDAVGWGFVVR GSKPCHIQAVDPSGPAAAAGMKVCQFVVSVNGLNVLHVDYRTVSNLIL TGPRTIVMEVMEELEC).
As used in the invention, the term “tumor marker” refers to is a substance that can be elevated in cancer, among other tissue types. An elevated level of a tumor marker may be used to indicate cancer. There are many different tumor markers, each indicative of a particular disease process, and they are used in oncology to help detect the presence of cancer.
According to the present invention, DEPDC6 protein is over-expressed in HCC tumorous tissue and its expression is elevated in colon and breast cancers and the tumor tissues from rectum, kidney and pancreas (see, for example,
In another aspect, this invention features a monoclonal antibody against DEPDC6 protein. In one embodiment, the monoclonal antibody is used in the detection of DEPDC6 protein. In a further embodiment, the monoclonal antibody is used to detect the expression level of DEPDC6 protein in human tissue. In another embodiment, the human tissue is isolated from an organ selected from the group of liver, colon, breast, rectum, kidney, pancreas and stomach.
Further, the present invention relates to a method to detect the presence or expression level of DEPDC6 protein in sample tissue by using a monoclonal antibody against DEPDC6 protein. In a further embodiment, it is provided a kit for detecting the presence or expression level of DEPDC6 protein in sample tissue, which is characterized by comprising a monoclonal antibody against DEPDC6 protein.
Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. Further, any mechanism proposed below does not in any way restrict the scope of the claimed invention.
To identify proteins that interact with GNMT, we used full length human GNMT as bait in a yeast two-hybrid screen system with a human kidney cDNA library. Human GNMT cDNA was subcloned into the pGBKT7 vector. A human kidney cDNA library fused to a pACT2 vector served as prey. The DEPDC6 carboxyl terminus (including its PDZ domain) was tested for interaction with GNMT. SV40 T antigen combined with p53 served as positive control. Yeast was grown in media lacking tryptophan and leucine (-W-L), which selects for the presence of plasmids. Only those combinations displaying a strong interaction grew under stringent conditions (i.e., media lacking tryptophan, leucine, adenine and histidine, or -W-L-A-H). According the results, the carboxyl-terminal region of a novel protein named DEP domain containing 6 (DEPDC6) was isolated (
Interactions of DEPDC6 with GNMT were also confirmed by co-immunoprecipitation (
We detected FRET between GNMT and DEPDC6 (n=10, 67%±4% efficiency), but not between GNMT and the EGFP control (i.e., zero FRET efficiency). We constructed eight plasmids containing different domains of either DEPDC6 or GNMT to determine specific domains responsible for their interactions using FRET-AB technique (
To generate rabbit anti-DEPDC6 antiserum and monoclonal antibodies against DEPDC6, purified GST-DEPDC6 or His-DEPDC6 were mixed with Freund's complete (for the initial immunization) or incomplete (for the booster injections) adjuvant (Sigma Co., St. Louis, Mo., USA) and the resultant mixture was used as an immunogen. His-DEPDC6 RP was used as a screened antigen for antibody arose by GST-DEPDC6 RP, and vice versa.
Mouse mAbs were produced by hybridoma technique (Liu et al., J Biomed Sci 2003; 10:87-97). The hybridomas were dispensed into six 96-well plates and cultured in a HAT medium (Chu et al., Hybridoma 1993; 12:417-417). The culture supernatants were screened using enzyme immunoassay (EIA) with GST-DEPDC6 RP and His-DEPDC6 RP. Hybridoma cells with high optic density were confirmed with Western blot assay immediately. Each well of cells with positive results were subcloned into a 96-well plate with a cell density of 0.5 cell per well. The resultant single clone with positive results were inoculated at a dosage of 5×106 to a BALB/c mouse which has been primed with 0.5 ml pristine (Sigma-Aldrich) previously. Monoclonal antibodies are purified from the mouse ascites with protein-A antibody purification kits (Pro-Chem Inc. Acton, Mass.) and concentrated using Centricon Plus-80 columns (Millipore). The isotype of each mAb was determined using a commercial kit (SouthernBiotech, Birmingham, Ala.).
High-tittered mAb purified from ascites were diluted with 0.1M NaHCO3 (pH 8.6) to a concentration of 100 μg/ml, and added to 6 ml sterile polystyrene Petri dishes. After coating overnight at 4° C. in a humidified container, the plates were blocked with the blocking buffer (0.1 M NaHCO3 pH 8.6, 5 mg/ml BSA, 0.02% NaN3, with a sterilized filter, stored at 4° C.) and incubated for at least 1 hour at 4° C. M13 phages displaying random heptapeptides at the N-terminus of its minor coat protein (pIII) were subsequently added (Ph.D.-7TM Phage Display Peptide Library, New England Biolabs Inc.). The phages bound to the plates were selected and repeatedly screened for 3 times before they were subjected to DNA sequencing. Detailed procedures have been published previously (Cortese et al., Curr Opin Biotechnol. 1996; 7:616-621). The hybridoma producing the mAb-D5 of the present invention was deposited with the German Collection of Microorganisms and Cell Cultures, on Jul. 4, 2012, as Deposit No. DSM ACC3180.
As shown in
Next, we tested whether the mAb D5 can be used in immunoprecipitation (IP) experiments. Mouse liver lysates and HuH-7 cell lysates were used for IP experiment. Lysates were incubated with 10 μg of mAb D5 for 1 h at 4° C., followed by the addition of 20 μl protein-A/G sepharose (Calbiochem, Merck KGaA, Darmstadt, Germany) and incubation for 4 h. The beads were washed three times with lysis buffer and resuspended in sample buffer for SDS-PAGE and Western blot analyses. The result showed that mAb D5 can precipitate endogenous DEPDC6 from both human HCC cell lysate and mouse liver lysate (
As shown in Table 1, the heptapeptides displayed at the N-terminus of the phage pIII protein were deduced by sequencing. More than three of isolated phages displayed a major sequence (consensus sequence) that could be used to identify the reactive epitopes for D5. When the consensus sequences were aligned with the human DEPDC6 protein sequence, the best matched regions spanned amino acid residues 245-251 (Table 1).
245-P
—a
aresidues appeared as conservative amino acids were indicated as dash marks
To determine DEPDC6 and GNMT expression levels in different HCC clinical stages or subgroups, we used real-time PCR to analyze 123 paired tumor (T) and tumor-adjacent (TA) tissues from HCC patients (Table 2). Compared to the TA tissues, the DEPDC6 expression level was significantly higher in the tumor tissues (p=0.008) (
Also note that DEPDC6 expression levels in tumorous tissues from patients with TNM stage I (but not in patients beyond TNM stage I) were significantly higher than those in the TA tissues (Table 3). This may be a result from the elevated DEPDC6 expression level in TA tissues observed in late stage patients. In contrast, GNMT expression levels in tumorous tissues were significantly lower than in TA tissues (p<0.001) (Table 3).
Diminished GNMT expression in HCC tumor tissues was consistently found in various patient sub-groups. An anti-DEPDC6 monoclonal antibody (D5) was used in immunohistochemical staining. Among the 51 pairs of tumorous and TA samples that were tested, 88% had either higher or equal expression levels of DEPDC6 in tumorous tissues compared to TA tissues (Table 4).
Slides of HCC patient tissues were incubated with monoclonal antibodies against DEPDC6. As shown in
Since DEPDC6 is frequently up-regulated in human HCC, we tried to determine if its overexpression enhances the transformational potential of cells. First, we compared the growth of HEK293T cells transfected with DEPDC6-expressing plasmid DNA with those transfected with a vector plasmid, and recorded cell doubling times of 19.5 h and 28.8 h, respectively (
To elucidate possible DEPDC6 functions, we used phylogenetic tree analysis with a neighbor-joining program to study evolutionary relationships among DEP domains from 11 proteins. DNA sequences from the following DEP domains were used to perform neighbor-joining phylogenetic analyses: DEPDC6 (GI: 189571663), P-Rex2 (also known as DEPDC2, GI: 47578114), PIKFYVE (GI: 121583482), RAPGEF4 (also known as EPAC2, GI: 155030205), GPR155 (also known as DEPDC3, GI: 74315999), DEPDC5 (GI: 55749916), DVL1 (GI: 32479520), RGS7 (GI: 156627562), PLEK1 (GI: 156616272), DEPDC1B (also known as BRCC3, GI: 23510330), and DEPDC4 (GI: 90855782). Bootstrap values greater than 700 from 1,000 replicate trees were considered significant (indicated at the nodes of corresponding branches). Results show that (a) the first DEP domain (DEP 1) of DEPDC6 clustered with the second DEP domain (DEP2) of P-Rex2 (also known as DEPDC2) (bootstrap 90%), and (b) the DEP2 domain of DEPDC6 clustered with Dishevelled 1 (DVL1) (bootstrap 86%) (
Furthermore, P-Rex2 serves as a link between Rac activation and the PI3K pathway, and its tandem DEP domains interact directly with the carboxyl-terminal region of mammalian target of rapamycin (mTOR). We therefore performed a pull-down assay to test whether DEPDC6 also interacts with mTOR. HEK293T cells were transfected with pHA-DEPDC6 and harvested for co-immunoprecipitation experiments. Cell lysates were incubated with anti-HA antibodies and precipitated using protein A/G beads. Immunoprecipitants were resolved on SDS-polyacrylamide gels and analyzed by immunoblotting. A specific interaction was observed between endogenous mTOR and DEPDC6, but not in the control experiment. As shown in
mTOR is a highly conserved protein kinase located downstream of PI3K and Akt. Its activation is associated with cell growth, survival, and proliferation. mTOR is found in two functionally and structurally distinct complexes, labeled mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The dysregulation of mTORC 1 signaling is frequently observed in human cancer tissues. In HEK293T cells, we observed that the transient overexpression of DEPDC6 blocked the phosphorylation of ribosomal protein S6—the downstream target of the mTOR signaling pathway (
Since the DEP2 domain of DEPDC6 shares high sequence homology with DVL 1, we used a T-cell factor/lymphoid enhancer factor (Tcf/Lef) luciferase reporter assay to determine the effects of DEPDC6 on the Wnt/β-catenin pathway. HEK293A cells were co-transfected with TOPFLASH reporter (950 ng), pRL-TK vector (50 ng, to control for transfection efficiency), and pHA-DEPDC6 (50, 200, and 800 ng). pcDNA3 plasmid was added to make total DNA amounts equal. At 36 h post-transfection, cells were serum-starved for 24 h prior to treatment with L-Wnt3a CM for 6 h. Cell lysates were measured for luciferase activity, expressed as arbitrary units relative to the activity observed in unstimulated cells normalized for Renilla luciferase activity. Experiments were performed in triplicate; error bars represent S.D. According to our results, reporter activity induced by L-Wnt3a conditioned medium (CM) was dose-dependently downregulated by DEPDC6 (
A further experiment was preformed to find out the effects of DEPDC6 when the pathway was activated via β-catenin overexpression, HEK293A cells were co-transfected with TOPFLASH reporter (950 ng), pRL-TK vector (50 ng, to control for transfection efficiency), pHA-(3-catenin (50 ng), and pHA-DEPDC6 (50, 200 and 800 ng). pcDNA3 plasmid was added to establish equal amounts of DNA. Cells were harvested at 36 h post-transfection for luciferase activity measurements. In contrast to the results observed in L-Wnt3a treatment, as shown in
The Wnt signaling pathway plays an important role in embryonic development, regulating cell proliferation and cellular polarity and determining cell fate 19. When the Wnt ligand binds to Frizzled receptor its signals are transduced to DVL, followed by the inactivation of β-catenin degradation complexes. The stabilization of β-catenin supports its association with T-cell factor (Tcf) in cell nuclei and activates targeted genes. According to the observations described above, DEPDC6 is significantly up-regulated in human HCC (
Since the DEP2 domain of DEPDC6 shares high sequence homology with DVL1, we used a T-cell factor/lymphoid enhancer factor (Tcf/Lef) luciferase reporter assay to determine the effects of DEPDC6 on the Wnt/β-catenin pathway. According to our results, reporter activity induced by L-Wnt3a conditioned medium (CM) was dose-dependently downregulated by DEPDC6 (
More intriguing, our data indicate that DEPDC6 may enhance β-catenin-stimulated Tcf/Lef-dependent transcription activity via crosstalk between Akt and the Wnt pathway. It has been demonstrated that deregulation of Wnt pathway is implicated in the pathogenesis of liver cancer and mutation of components within β-catenin destruction complex, such as APC and AXIN, are common in many types of cancer including HCC. We previously used a Tcf/Lef reporter assay to demonstrate that (a) GNMT inhibits Wnt signaling, and (b) cyclin D1 and c-myc (target genes of Wnt signaling) are up-regulated in the adult livers and HCC tissues of GNMT −/− mice, meaning that both GNMT and DEPDC6 play important roles in PI3K/Akt/mTOR and Wnt/β-catenin pathways.
In the model shown in
Additionally, we detected the expression of DEPDC6 in various organs by IHC staining. Monoclonal Ab-D5A was used to detect expression profile of DEPDC6 in different organs. Multiple cancer tissue array (US Biomax, Cat. No. BCN721) was incubated with monoclonal antibody D5A. Signals were visualized using SuperPicTure™ Polymer Detection kits (Zymed, Invitrogen, Carlsbad, Calif., USA).
As shown in
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
