Abstract Pancreatic cancer (PCa) typically is associated with a poor prognosis and a shortage of molecular therapeutic targets. Notch-1 belongs to the Notch family of transmembrane receptors and is predominantly expressed in Pca, and the mechanism for its activation has not been elucidated. In search for the regulatory mechanisms of Notch-1 receptor intracellular domain (Notch-1ICD), the activated form of Notch-1 receptor, we investigated whether c-Src plays a role in the regulation of Notch-1 expression in PCa HPAC cells. Here we reported that transforming growth factor alpha (TGFα) treatment induced the Notch-1ICD protein expression almost immediately in a dose and time dependent manner and the activation was partialy abrogated by the the Src-family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-d] pyrimidine (PP2) with Western blotting. Blockade of c-Src with PP2 and using siRNA to knock-down c-Src proteins, we found that the protein expression of Notch-1 ICD is c-Src-dependent. Data from Co-immunoprecipitation(Co-ip) revealed that endogenous Notch-1 could physically interact with c-Src in PCa HPAC cell lysates and in overexpression system. We performed confocal scanning laser microscope and demonstrated that the colocalization of c-Src with Notch-1 in PCa HPAC cells, human PCa tissue sample and nude mice xenografted tumor. Expression of various mutant Src constructs, the additional studies demonstrated that Notch-1 independently interact with the c-Src KD domain. Data from MTT assay suggested that overexpression of Notch-1 intracellular domain resulted in the reversion of the cell proliferation inhibition promoted by PP2. Taken together, there may exsit a c-Src mediated Ligand-independent activation of Notch-1, and the underlying mechanisms at least in part by direct protein-protein interactions between Notch-1and c-Src.
Introduction
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PCa is one of the most aggressive human malignancies, and has a very poor prognosis (1-2). Notch signalling is pivotal for regulation of cell fate and growth. Aberrant activation of the Notch pathway is commonly observed in pancreatic cancer (3-6), but the mechanism for its activation has not been elucidated. There are four Notch members (Notch-1-4) have been identified among vertebrates. These show considerable structural homology within their intracellular regions and share common domain architectures, including the RAM (RBP-Jκ-associated molecule) domain, the seven ankyrin repeats (ANK) domain, and a carboxy-terminal PEST sequence (7). Notch activation is always ligand dependent in PCa. Synthesized Notch receptors are proteolytically cleaved during translocate to the cell surface, creating heterodimeric mature receptors comprising noncovalently associated extracellular (NEC) and transmembrane (NTM) subunits. On binding to ligands, the Notch receptor undergoes two proteolytic cleavages, catalyzed by metalloproteases and gamma-secretase, respectively. The Notch intracellular domain (NotchICD) is released as the same time translocate to the nucleus and interacts with CSL factors and transcriptional co-activators to modulate the expression of target genes (8). However, the precise molecular mechanism by which Notch-1 is activated needs additional investigation. c-Src is the most closely related members of the Src family of nonreceptor tyrosine kinases, and displays a common modular structure consisting of the unique, SH3, SH2, and catalytic domains (9). Up-regulation of c-Src correlates with a variety of human tumors, including cancer of the pancreas (10-11). Obstruction of c-Src signaling results in significant inhibition of cacer cell proliferation, invasion and marked induction of cell apoptosis. (12-14). Since c-Src and Notch-1 together have been found to be overexpressed in human pancreatic cancer, whether the ubiquitous c-Src participate in the Notch-1activation still remains to be investigated. In search for the regulatory mechanisms of Notch-1 in PCa, we investigated whether regulation of c-Src protein level could mediate the expression of Notch-1receptor, by using small interfering RNA (siRNA) and PP2, which is a pharmacological inhibitor of Src kinase. Our results suggest that down-regulation of c-Src caused decreased expression of Notch-1, resulting in the inhibition of pancreatic cancer cell growth mediated through the cross-talk between the two key protein molecules. Our research also hint that hence targeting the c-Src mediated Notch-1- signaling pathway might be a promising strategy for developing chemopreventive agents against Pancreatic cancer.
Materials and Methods
Cell culture and experimental reagents
Human pancreatic cancer cell lines HPAC and BxPC-3 were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and1% penicillin and streptomycin. All cells were cultured in a 5% CO2 humidified atmosphere at 37℃. Primary antibodies for Notch-1, c-Src, β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). All secondary antibodies were obtained from Pierce (Rockford, IL). goat anti-rabbit Alexa Flour 568 (Molecular Probes, Eugene, OR) or goat anti-mouse Alexa Flour 488 (Molecular Probes). c-Src small interfering RNA (siRNA) and control siRNA were obtained from shanghai GenePharma SiRNA company. Lipofectamine 2000 was purchased from Invitrogen(Invitrogen Corp., Carlsbad CA). Chemiluminescence detection of proteins was done with the use of a kit from Amersham Biosciences (Amersham Pharmacia Biotech). Protease inhibitor cocktail, A/G Agarose beads were obtained from Santa Cruz Biotechnology (Inc. Santa Cruz, CA). 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) were obtained from (Enzo Life Sciences International, USA), Protease inhibitor cocktail, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Solarbio, China), monoclonal anti-hemagglutinin (HA) and anti-flag antibody (Applygen Technologies Inc. China), and all other chemicals were obtained from Sigma (St. Louis, MO).
Plasmid construction and transfection
Human c-Src was cloned by RT-PCR from human pancreatic cancer total RNA and inserted into a HA-tagged mammalian expression vector, pcDNA3 vectors. The c-Src and mutants, SH3, SH2 and KD were generated by PCR amplification and inserted into the same vector. The human NICD was cloned by RT-PCR from human pancreatic cancer cell total RNA and inserted into the pcDNA3-FLAG vector. The constructs for the NICD mutants were generated by PCR amplification and cloned into the same vectors. The identities of all constructs were verified by DNA sequence analysis. Transfections were performed with Lipofectamine 2000 reagent (Invitrogen Corp., Carlsbad CA) according to the manufacturer's instructions.
Small Interference RNA
SiRNA designed against the region of the human c-Src gene were synthesized .c-Src siRNA sequence as follow, sense strand：5'-CAAGAGCAAGCCCAAGGAUtt-3', Anti sense strand 5'-AUCCUUGGGCUUGCUCUUGtt-3'；Control siRNA sequence as follow, sense strand：5'-UUCUCCGAACGUGUCACGUtt-3', Anti sense strand：5'-ACGUGACACGUUCGGAGAAtt-3'. HPAC cells (1.5 - 105) were transfected with siRNA at a final concentration of 50 nmol/L using Lipofectamine 2000. At 48 h after transfection, cell lysates were extracted for Western blotting to assess relative c-Src and Notch-1 protein level.
Western Blot Analysis
Cells were lysed in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA, 100 mM NaF, and 1 mM Na3VO4) containing protease inhibitor cocktail, by incubating for 20 minutes at 4℃. The protein concentration was determined with the BCA method (Pierce, USA). Total proteins were electroblotted to PVDF membrane using a wet transblot system (Bio-Rad, Hercules,CA). Blots were then blocked for 1 h at room temperature with10% bovine serum albumin (BSA) or 5% nonfat dry milk in phosphate-buffered saline tween-20 (PBST). After three 5min washes with PBST, membranes were incubated overnight at 4°C with antibodies to Notch-1, c-Src and β-actin diluted 1:1,000 in PBST. After subsequent washes, the membranes were incubated for 1 h with horseradish peroxidase conjugated goat anti-rabbit or anti-mouse, diluted 1:5,000 in PBST. After washing, the membrane was processed using Super Signal West Pico chemiluminescent substrate (Pierce, USA), followed by exposure to Fujifilm LAS3000 Imager (Fuji, Japan). The results were evaluated with densitometric analysis by using Image J Analyst software (NIH, USA).
Coimmunoprecipitation
HPAC cells were washed with ice-cold PBS and lysed in 1 ml of RIPA buffer for 30 min on ice, then clarified by centrifugation. 1 mL cell lysate (1 mg/ml), and 10μL Notch-1, c-Src (conc: 200μg/mL) , 2μL IgG (conc: 1μg/μL) antibody were incubated overnight at 4 ℃. 50μL of protein A/G PLUS-agarose was added and mixed at 4℃ for 2 h with gentle agitation. The pellet was washed three times with 50μL 2-loading buffer (Tris pH 6.8, 0.1% SDS, 10% glycerol, and 0.025% Bromphenol blue, 20 mM DTT) was heated at 95℃ for 5 min and the pellet fraction centrifuged 12000 for 5 min prior to gel loading and Western blot analysis. Hela cells (2-104) were transfected with 4μg of the indicated Flag-NICD and HA-Src plasmids using Lipofectamine 2000. After 24 h, cells were rinsed with ice-cold PBS and resuspended in 1ml of RIPA buffer. The pre-cleared lysate was then incubated for 1h at 4℃ with the Flag or HA antibody and the resulting immune complexes were collected on protein A/G PLUS-agarose beads. Immune complexes were then captured by centrifugation, washed extensively in lysis buffer and solubilized with 2-loading buffer before loading onto a SDS-PAGE gel.
Confocal microscopy
HPAC cells were plated at a low confluency of ~60% on a 12-mm-diameter glass coverslip in six-well plates (Corning, NY). After 24 h, cells were washed three times with PBS, and fixed in 4% formaldehyde for 20 min at room temperature. Following a wash with PBS, cells were blocked with 5% BSA in PBS for 1 h at 37℃ and incubated with a 1:100 dilution of rabbit anti-Notch-1or mouse anti- c-Src antibody (Santa Cruz) for overnight at 4°C on a rocking platform. Washed slides were incubated for 1 h at room temperature with 1:150 dilutions of FITC conjugate goat anti-mouse IgG (H+L) for Notch-1 or CY3 conjugate affinipure goat anti-rabbit IgG(H+L) (1:150) secondary antibody for c-Src. Slides were then washed and mounted on a glass slide with a mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (catalog no. ZLI-9057, Zhongshan Goldenbridge). The slides were stored in the dark at- 20°C until they could be examined using a confocal microscope (Leica Microsystems, LAS AF-TCS SP5). Negative controls, designed to assess non-specific binding, were conducted in parallel. For these controls, cells were treated as described above except that they were not exposed to primary antibody. Merging red and green showed colocalization of two proteins giving yellow color. Hela cells (5-105/well) were seeded seeded on a 12-mm-diameter glass coverslip, placed in a 6-well plate and transfected with 0.4μg of the indicated Flag-NICD and HA-Src plasmids using Lipofectamine 2000, and processed for confocal microscopy to evaluate the protein colocalization.
HPAC xenografts
Four-week-old female BALB/cA-nude mice were obtained from Animal Laboratory of Capitial Medical University and allowed free access to food and water. All animal experiments were carried out with the approval of the ethical committee. By i.p. injection into the flank area, nude mice were inoculated with 107 HPAC cells (in serum-free RPMI 1640) in a total volume of 0.1 mL of PBS. Tumor volume was estimated as previously described (15). After inoculation, mice were randomized to two groups: (a) treatment group (n =6) received 2 mg/kg PP2 in 1% DMSO by i.p. injection three times a week; (b)The control group received the same volume of 1% DMSO vehicle as the first groups, three times a week. At the time Tumor tissues were harvested for immunofluorescence analysis.
Cell growth inhibition studies by MTT assay
The cell was seeded at a density of 5,000 cells per well with 100μl medium in 96-well plate and then transfected with FLAG-NICD (2μg) and negative vecror (2μg) respectively, as described above. Dimethyl sulfoxide (DMSO) only and Soluble PP2 in DMSO were added to each well in different concentration (0μM, 5μM, 10μM and 20μM), and the MTT assay was performed after 24h post transfection. Additionly, 10μM of PP2 was added to each well and the MTT assay was performed at 24, 48, 72, and 96 h post transfection. 20μl of MTT reagent (5mg/mL) was added to each well, and incubated in a 5% CO2 humidified atmosphere at 37℃for 4h. After removing the medium, 200μl DMSO was added to each well to dissolve the formazan and the absorbance was measured at 490 nm.
Densitometric and Statistical Analysis
The bidimensional absorbances of Notch-1, c-Src and β-actin proteins on the films were quantified and analyzed with Image J Analyst software (Bio-Rad). The ratios of Notch-1, c-Src and β-actin were calculated. The MTT assay experiment was performed in triplicates. Data represent means ± SE from three independent experiments. Statistical analysis was performed by Student's t-test or ANOVA for analysis of variance when appropriate (P < 0.05 was considered significant).
TMA immunohistochemical study
Paraffin wax embedded tissue from 30 cases of pancreatic cancer and 10 cases of normal Pancreatic tissue was studied by confocal microscopy with a 1:100 dilution of rabbit anti-Notch-1or mouse anti- c-Src antibody, and incubated with 1:150 dilutions of FITC conjugate goat anti-mouse IgG for Notch-1 or CY3 conjugate affinipure goat anti-rabbit IgG secondary antibody for c-Src. For negative control, the slides were treated with PBS in place of primary antibody. More than 10 visual fields were observed or more than 1000cells were counted per sample. The positive index of Notch-1and c-Src represented the expression of protein. The positive colocalization index of Notch-1and c-Src = number of positive cells/1000 cells. The number of positive cells was recorded with Image-Pro Plus® Version 5.1.0 software (Media Cybernetics Inc).
Results
Effects of Src inhibitor PP2 on TGF alpha-induced Notch-1 activation in PCa HPAC and BxPC3 cells
To investigate whether c-Src kinase is involved in regulation of Notch-1 activation, Src kinase inhibitor PP2 were tested. After PP2 treatment for 30 min, we found it significantly reduced the protein expression level of 120kDa Notch-1 for more than 2 folds. The PP2-induced Notch-1 protein inhibition seems to be dose and time- dependent (Fig.1A) in PCa HPAC and BxPC3 cell lines. Our results show that 10uM PP2 treatment for 30 min did change Notch-1 protein expression. To role out that PP2-induced inhibition of Notch-1 protein expression is chemical structure-dependent, we also tested effect of SU6656, which belongs to different structual class of Src kinase inhibitors. We found SU6656 had similar effects as PP2 (Fig.1B).
Transient transfection of c-Src-specific siRNA suppresses Notch-1 expression
While the above experiments suggested that PP2 alone suppressed the protein expression of Notch-1 in a time-course and dose-response -dependent manner. To confirm further, we next applied siRNA-mediated gene knock-down. The effect of c-Src-specific siRNA treatment on c-Src and Notch-1 expression were examined .As shown in Fig. 2, the expression of c-Src was clearly abolished and Notch-1 protein expression was reduced by-50% synergistically in HPAC cells treated with a mixture of modified siRNA directed against c-Src, whereas a control siRNA mixture had no detectable effect despite transfection efficiencies of -80% in both instances. These results strongly suggest the involvement of c-Src in Notch-1regulation in PCa HPAC cells.
NICD overexpression reduced Src inhibitor PP2 induced cell growth inhibition by using a MTT assay (Time-course and dose-response)
HPAC cells treated with 0 μmol/L, 5 μmol/L, 10 μmol/L, and 20 μmol/L PP2 for 24 hours, 48 hours, 72 hours and 96hours.The control lane depicts cells that were treated with DMSO. The results indicated that the treatment of HPAC pancreatic cancer cells with PP2 resulted in cell growth inhibition, and the inhibition of cell growth was dose-dependent and time-dependent. HPAC cells were transfected with NICD, grown to confluence, and the effect of PP2 on cell proliferation was measured as just described. As shown in Fig. 6, overexpression of NICD significantly increased growth of HPAC cells (p=0.01) compared to empty plasmid transfection, suggesting that overexpression of NICD completely counteracted the inhibitory effect of PP2 on cell growth inhibition bringing cell proliferation.These experiments strongly suggest that inhibiting c-Src kinase activity decreased cell growth in HPAC cells, and overexpressing NICD increased cell growth .
c-Src directly binds to Notch-1 and NICD in vivo
Even though there is a report showing that the p56lck.Notch-1 complex is present in primary T cells [16]Sade H, Krishna S, Sarin A. The anti-apoptotic effect of Notch-1 requires p56lck-dependent, Akt/PKB-mediated signaling in T cells. J Biol Chem, 2004, 279:2937-44. It is unclear whether the binding between c-Src and Notch-1 may exsit and how affect Notch-1 activation in pancreatic cancer cells. First of all, we then did co-immunoprecipitation of c-Src and Notch-1 to see if c-Src are directly associated with Notch-1 in PCa HPAC cells. As shown in Fig.2A, we found that endogenous significant amount of c-Src and Notch-1 were specifically immunoprecipitated with the counterpart antibody for Notch-1 or c-Src in HPAC cells, respectively. Confocal study shows that c-Src was co-localized with Notch-1 in PCa HPAC cells. (Fig.2 B). The results suggestsed that endogenous c-Src may physically associate with Notch-1 in vivo.
Although the interaction between c-Src and Notch-1 was apparent in a cellular context, it was unclear whether the two proteins bind directly or not. To further confirm the in vivo interaction between c-Src and Notch-1, we performed co-immunoprecipitation overexpression experiments. HA-tagged c-Src and FLAG-tagged NICD expression plasmids were introduced into HeLa cells. The cell lysates were precipitated with either control IgG or anti- FLAG monoclonal antibody, and the precipitated complex was detected for the presence of HA- c-Src by western blotting using anti-HA monoclonal antibody. The cell lysates were precipitated with either control IgG or anti- HA monoclonal antibody, and the precipitated complex was detected for the presence of FLAG- NICD by western blotting using anti-FLAG monoclonal antibody, respectively, As shown in Fig. 3A, c-Src and Notch-1 could be detected in immune complexes precipitated by anti-HA and anti-FLAG monoclonal antibody, but not by IgG, indicating that c-Src can interact with NICD.Therefore, we determined whether these two proteins share the same location in a cell using immunofluorescence staining. Consistent with the localization of overexpressed HA-tagged c-Src, endogenous Notch-1 protein is localized in the HeLa cells (Fig. 3B). Interestingly, although both c-Src and Notch-1 are predominantly localized in the cytoplasm that close to nucleus, the protein signals are merged mainly in the Golgi apparatus (Fig. 3C), suggesting that the interaction of the two protein occurs mainly in trafficking.
In order to determine the binding domain of c-Src on the Notch-1, the immunoprecipitation assay was performed on a Notch-1 with the deletion constructs of c-Src. c-Src consists of an SH3domain of 130 amino acids, SH2 domains constituted of 42 amino acids, and a carboxy-terminal domain of 100 amino acids. As a result of immunoprecipitation, Notch-1 was able to bind the KD deletion constructs of c-Src (Fig. 4). Together, these data provide the first biochemical evidence that c-Src and Notch-1 interact each other directly, and a central kinase domain of c-Src is essential for the interaction between c-Src and Notch-1.
Toward this goal, a prostate tissue microarray block containing 432 tissue cores (0.6 mm diameter) was constructed.
Notch-1may be tyrosine phosphorylated by c-Src
Above studies have shown that Src family PTKs c-Src can interact with the Notch-1 receptor. To test whether Notch-1 receptor were tyrosine phosphorylated, we prepared PCa HPAC cells lysates, immunoprecipitated individual Notch-1, and analyzed them for phosphotyrosine content by immunoblotting using anti-phosphotyrosine antibody 4G10. As shown in Figure 5, Notch-1 were tyrosine phosphorylated. There are several tyrosine residues in the intracellular regions of the Notch-1 receptor that are potential tyrosine phosphorylation sites[17]. Using immunoprecipitation experiments, we estimated that 1-2% of Notch-1 was tyrosine phosphorylated in the human PCa HPAC lysate (data not shown).
Immunofluorescence analysis of PCa tumor samples
We next investigated the relationship between c-Src and Notch-1 expression level in clinical PCa samples by immunofluorescent staining of TMA. Representative images are presented for c-Src and Notch-1 in Fig.7. Cytoplasmic/nuclear staining of c-Src was observed in 126 of the 30PCa samples. Notch-1 staining with polyclonal antibody specific to Notch-1 was mainly observed in the cytoplasm of tumor cells. We regarded strong staining as a positive result for DKK1 and cytoplasmic/nuclear staining as a positive result for β-catenin (Table S1).
Conclusion
The major findings of the present study were that non-receptor tyrosine kinase c-Src may play a role in the regulation of Notch-1 expression in PCa HPAC cells, the activation of Notch-1 at least in part by direct protein-protein interactions between Notch-1and c-Src. Some studies have revealed that aberrant Notch signaling inhibited cell differentiation and caused tumors (1, 2), and it has been observed in human PCa tissue samples (3, 4), many PCa cell lines (5, 6), and animal models (7, 8). With regard to human Notch, the story has become more complicated. Focused on Notch-1, previous studies have showed that the up-regulation of Notch-1 may repress the differentiation of tumor-initiating cells in PCa (18, 19). Our result also indicate that Notch-1 has a increased cytoskeletal distributions in HPAC cells and subcutaneous tumors compared to control group. However, that Notch was not sufficient for tumorigenesis when it was activated alone (21) but needed to synergize with additional molecular alterations to promote neoplastic transformation (Leong and Karsan, 2006). The cross-talk between Notch and other oncogenetic signaling pathways could then form a feed-forward loop that promoted tumor cell growth (Girard et al., 1996; Palomero et al., 2006). c-Src is originally viewed as the representatives for nonreceptor tyrosine kinases. Substantial evidence implicates that c-Src has been verified as an important mediator in cellular functions such as tumorigenesis, cell migration, and cell survival. To date, a variety of proteins have been identified and characterized as the substrates for c-Src (23, 24). Among the numerous c-Src targets, we detected whether Notch-1 receptor is the one that participate in will be described below.
Our results also demonstrate that (40), upon stimulation by TGF-a, Notch expression are activated. Treatment with the c-Src conventional specific inhibitors PP2 or SU6656 caused strong inhibition of Notch-1 activation. These results suggest a strong rationale for inhibiting endogenous c-Src expression to inhibit constitutive Notch-1 activation. To further clarify the role of c-Src mediate Notch-1expression in pancreatic cancer development, additional experiments in which c-Src was either knocked down or using pharmacologic inhibitors of PP2 in different concentration and time cource. As demonstrated in Figure 5B, knockdown or inhibition the expression of c-Src diminished the expression of Notch-1. That suggest Notch-1 generally produces a synergetic effect on the expression of c-Src protein, and that its effects work through a c-Src-dependent mechanism. Our data also show that down-regulation of c-Src reduced cell growth in vitro and subcutaneous tumors growth in HPAC xenografts in vivo, but overexpression of NICD can reverse the HPAC cell growth inhibition induced by PP2. The study suggest that c-Src may stimulate pancreatic tumor cell proliferation by mediating the activation expression of Notch-1 receptor.
The mechanism has not been elucidated that c-Src might possibly regulate the activity of Notch-1. In this study, we identified c-Src as a novel binding partner of Notch-1. The interactions between c-Src and Notch-1 were verified by co-immunoprecipitation experiments in HPAC cells. As shown by the immunofluorescence study, endogenous c-Src co-localized with Notch-1 in the cytoplasm of the HPAC cells, xenografts nude mice and PCa TMA. Based on these results taken together, one of the major conclusions from the present work is that c-Src physically associate with Notch-1 in PCa. Utilizing the deletion mutant expression vector series for c-Src, we determined that the SH2 region of c-Src and NICD were responsible for the interactions. We speculate that, other Src family members may also be involved in Notch-1 receptor regulation procession in addition to Lck(4)and c-Src. Addtionally, we report that c-Src is expressed not only in the membrane and cytoplasm, but also in the nuclei of human PCa HPAC cells and subcutaneous tumor cells. Our study also find that c-Src inhibitor PP2 reduced the nuclear expression of c-Src and decreased the localization of c-Src and Notch-1protein mechanistically at the same time. Such as the previous study described, at the cellular level, Notch/Kras coactivation promotes rapid characteristically oncogenesis (30).Overall, this biological synergy between the c-Src and Notch-1may associated with the formation of a heterocomplex between these two protein.s
Previously published data have suggested that c-Src inhibitors are promising treatment for gastric cancer [18]. Downregulation of Notch can induce apoptosis in PCa [28,29]. Thus, our discovery of Notch-1 pathways regulated by c-Src makes it an attractive target for therapeutic intervention. In conclusion, we provide here for the first time that c-Src could regulate Notch-1activition at the protein level in PCa and the up-regulation is largely dependent of the binding of the two molecula mechanismly. An extensive understanding of Notch signaling cascade and its interaction with other pathways has provided us with insightful information for the identification of molecular targets to design effective therapeutic strategies. Future studies should elucidate the mechanism of, and requirement for, interaction between these critical signaling pathways.
[21]Ayyanan A, Civenni G, Ciarloni L, et al. Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism.Proc Natl Acad Sci U S A,2006,103:3799-804
[28] Wang Z, Azmi AS, Ahmad A, Banerjee S, Wang S, Sarkar FH, et al. TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and induces apoptosis in pancreatic cancer: involvement of Notch-1 signaling pathway. Cancer Research 2009;69:2757-65.
(29)Wang Z, Zhang Y, Li Y, Banerjee S, Liao J, Sarkar FH. Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells. Mol Cancer Ther 2006;5:483-93.
(30) De La OJ, Emerson LL, Goodman JL, et al. Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia.Proc Natl Acad Sci U S A,2008,105:18907-12.
Conflict of interest statement
No potential conflicts of interest were disclosed.
1 A. Jemal, R. Siegel and E. Ward et al., Cancer statistics 2008, CA Cancer J Clin 58 (2008), p. 71. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (3921)
Acknowledgements
This work was funded by National Natural Science Foundation of PR China grants 30271448, 30471975, 30393113 and Education Ministry of P. R. China grant 03003.We thank Lan Yuan for assistance with confocal analysis. Correspondence and Requests for materials should be addressed to: Corresponding author at. Department of Biochemistry and Molecular Biology, Peking University Health Science Center,Xue Yuan Road 38, Beijing 100083, PR China. Tel.: +86 10 82801434; fax: +86 10 82801434.
