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Recurrence and subsequent metastatic transformation of cancer develops from a subset of malignant cells, which show the ability to resist stress and to adopt to a changing microenvironment. These tumor cells have distinctly different growth factor pathways and anti-apoptotic responses compared to the vast majority of cancer cells. Long-term therapeutic success can only be achieved by identifying and targeting factors and signaling cascades that help these cells to survive during stress. Both microarray and immunohistochemical analysis on human prostate cancer tissue samples have shown an increased expression of vascular endothelial growth factor-C (VEGF-C) in metastatic prostate cancer. We have discovered that VEGF-C acts directly on prostate cancer cells to protect them against oxidative stress. VEGF-C increased the survival of prostate cancer cells during hydrogen peroxide stress by the activation of AKT-1/PKBα. This activation was mediated by mTOR complex 2 (mTORC-2) and was not observed in the absence of oxidative stress. Finally, the transmembrane non-tyrosine kinase receptor Neuropilin-2 was found to be essential for the VEGF-C-mediated AKT-1 activation. Indeed, our findings suggest a novel and distinct function of VEGF-C in protecting cancer cells from stress-induced cell death, thereby facilitating cancer recurrence and metastasis. This is distinctly different from the known function of VEGF-C in inducing lymphangiogenesis.
The increased ability of tumor cells to survive under stress is important for cancer progression, subsequent metastatic transformation, and therapy resistance (1). The stress can be incurred by the microenvironment surrounding the cancer cells and/or by therapeutic interventions used to treat cancer (2). Alternative growth factor pathways and adaptive upregulation of antiapoptotic mechanisms are the primary causes for cancer cells to evade death during unfavorable conditions (3). Reactive oxygen species (ROS) and cellular oxidative stress have long been understood to be associated with cancer, although this association is often complex and paradoxical (4). Oxidative stress may induce cancer, and receptor tyrosine kinase activated cell cycle progression often involves an increase in ROS signaling (3, 5). On the other hand, the antioxidant system in cancer cells increases paradoxically as transformed cells generate higher level of ROS compared to normal cells (6). Indeed, several therapeutic agents promote cell death by increasing oxidative stress (7, 8). A number of agents, including radiotherapy, chemotherapeutic drugs like paclitaxel, histone deacetylase inhibitors, proteasome inhibitors, as well as redox cycling agents, increase oxidative stress (6, 9). This common effect suggests that cancer cells are more vulnerable to oxidant stress, because they function with an enhanced basal level of ROS-mediated signaling (10). Therefore, by further increasing the ROS level by these therapeutic agents cancer cells are pushed beyond the breaking point of damaging cellular organelle and DNA, and undergo apoptosis. Thus, a recurrence of the tumor after therapy likely results from a subset of cells that have developed the ability to overcome oxidative damage (3, 9). These cells also acquire a metastatic phenotype, which become the major cause of death due to the cancer (1, 2).
The tumor microenvironment, which is highly heterogeneous in terms of nutrient supply, pH and oxygenation, plays a major role in the ability of tumor cells to resist stress by altering gene expression and cellular functions of cancer cells (11, 12). The presence of inflammatory cells, myofibroblasts and endothelial cells in the tumor microenvironment supports not only the growth of tumor cells but also its ability to resist stress and therefore facilitates metastasis (11). The metastatic cascade is thus orchestrated by signals from both a tumor and its microenvironment. In this context, the growth factors and cytokines that facilitate communication between the tumor cells and the stromal compartment are particularly important. For instance, members of the vascular endothelial growth factor (VEGF) gene family are known to execute a functional communication between the tumor cell and its surrounding environment (13, 14).
VEGF-C appears to be particularly unique among the VEGF family members (15, 16) because of its involvement in the lymph node metastasis (17). A number of studies have reported a significant correlation between the expression of VEGF-C and lymph node metastases in human prostate carcinoma (18–21). The cancer microarray profile database, Oncomine, also describes a significant upregulation of VEGF-C mRNA in human metastatic prostate cancer tissue specimens (22, 23). VEGF-C functions by activating its cognate tyrosine kinase receptors VEGF-R3 (Flt4) (24) and VEGF-R2 (KDR), and the non-tyrosine kinase receptor neuropilin-2 (NRP-2) (25). These receptors were identified initially on lymphatic endothelial cells (26), and one of the known functions of VEGF-C is to promote the formation of new lymphatic vessels by inducing the proliferation, migration and sprout formation of existing lymphatic endothelial cells, a process called lymphangiogenesis (27). It has been postulated that, by inducing lymphangiogenesis, VEGF-C facilitates lymph node metastasis (28). Furthermore, several reports now point to functions for VEGF-C, which are independent of lymphangiogenesis, and instead are important for cancer progression. For example, VEGF-C can stimulate the proliferation and migration of Kaposi’s sarcoma cells and also the proliferation and survival of leukemia cells (29). Lack of lymphangiogenesis has been reported in uveal melanoma despite high VEGF-C expression (15). More importantly, VEGF-C is often overexpressed in glioblastoma patients; although brain tissue is void of lymphatics (30). In a recent report, it has been postulated that the risk of childhood neuroblastoma treatment failure (progression or relapse) as well as tumor related death was found to be significant in VEGF-C positive patients (31). VEGF-C has also been suggested to be a trophic factor for neural progenitors in the vertebrate embryonic brain (32). An autocrine function of VEGF-C to promote the invasion and metastasis of lung, breast and gastric cancers has also been reported (33–35).
In this study, we have identified a survival promoting function of VEGF-C on prostate cancer cells under severe oxidative stress conditions. We have also delineated the underlying molecular mechanism for this stress-resistant function of VEGF-C, which involves the activation of mTOR complex 2 (mTORC-2) and AKT-1. The non-tyrosine kinase VEGF-C receptor, neuropilin-2 was identified as an upstream component in this pathway. Our findings therefore provide a novel mechanism by which VEGF-C protects cancer cells from stress induced cell death. It also sheds new light on the upstream events of mTORC-2 activation.
Human prostate cancer cell lines LNCaP (ATCC # CRL-1740), LNCaP C4-2 (ViroMed Laboratories, Minnetonka, MN) and PC3 (ATCC # CRL-1435) were cultured at 37°C in in RPMI 1640 with L-glutamine (Mediatech Inc., Manassas, VA) supplemented with penicillin/streptomycin and containing either 10% Fetal Bovine Serum (Hyclone Laboratories, Fisher Scientific, Pittsburg, PA). Cells were serum starved overnight before adding recombinant wild type VEGF-C (R&D Systems Inc., Minneapolis, MN) for 9 hours. After 4 hours of VEGF-C incubation, hydrogen peroxide (Sigma-Aldrich, St. Louis, MO) at different concentration was added for another 5 hour.
Immunoprecipitation was performed with 0.75 mg of total cellularprotein from whole cell extracts with antibody(1 μg) directed against mTOR (Cell Signaling Technology, Danvers, MA) and pulled down by protein A-agarose beads (Pfizer-Pharmacia, New York, NY). Western blots were conducted using antibodies against phospho -Serine 473 AKT-1 (Upstate-Millipore, Billerica, MA), phospho Threonine 308 AKT, AKT-1, Phospho-GSK-3β, phospho-FOXO-1, 4EBP-1, S6, mTOR, rictor, phospho 4EBP-1, phospho-S6 (Cell Signaling Technology), GSK-3β (BD Biosciences, San Jose, CA), neuropilin-2 (R&D Systems Inc.), rho-GDI (SantaCruz Biotechnology, Inc., Santa Cruz, CA) and β-actin (Sigma-Aldrich).
Vybrant Apoptosis Assay Kit #7, purchased from Molecular Probes-Invitrogen Detection Technologies (Eugene, OR), was used according to the manufacturer’s protocol. Briefly, LNCaP and LNCaP C4-2 cells (2×106 cells/well, 6-well plate) were serum starved overnight followed by recombinant VEGF-C and hydrogen peroxide addition as mentioned in “Cell Culture” section of “Materials and Methods”. PC-3 cells were transfected with VEGF-C specific siRNA for 72 hours followed by hydrogen peroxide treatment for 5 hours. Adherent cells were washed with PBS were incubated with 1 μl each of Hoechest 33342 stock solution, YO-PRO-1 stock solution and propidium iodide stock solution at 37°C in an atmosphere of 5% CO2 for 15 minutes. The cells were viewed under a fluorescence microscope using appropriate filters. The apoptotic cells were stained with green-fluorescent YO-PRO-1 dye and the dead cells were stained with red-fluorescent propidium iodide dye. The blue-fluorescent Hoechest 33342 stains the chromatin of the cells.
Cells were transfected with siRNA for Neuropilin-2 (Santa Cruz Biotechnology, Inc.) and VEGF-C (Dharmacon RNA Technologies, Chicago, IL) as published using Dharmafect 2/3 (Dharmacon RNA Technologies). siRNA transfection was allowed to proceed 72 hours before collection of whole cell extract or total RNA.
Mature form of VEGF-C expressing plasmid (pSecTag2BΔNΔCVEGF-C, a kind gift from Dr. Mihaela Skobe) was transfected to LNCaP C4-2 cells. For vector-only control pSecTag2B plasmid with no insert was transfected in parallel to another set of LNCaP C4-2 cells. Cells were selected with Zeocin. Multiple stable clones of LNCaP C4-2 cell line expressing varying levels of VEGF-C will be isolated for subsequent experiments.
Receptors for VEGF-C have been characterized on a number of cancer cells (34, 36), suggesting the possibility of cancer cell-specific functions of VEGF-C. We have detected the VEGF-C receptor Neuropilin-2 in prostate cancer cell lines such as LNCaP, LNCaP C4-2 and PC-3 (Figure 1A). LNCaP C4-2 is a hormone refractory or castration recurrent prostate cancer cell and is more metastatic than its syngenic parental cell line LNCaP. This cell line was developed by isolating tumor cells from the regional lymph nodes after injecting LNCaP cells orthotopically into castrated mice (37). LNCaP C4-2 cells are not dependent on androgen for their growth, even though they express androgen receptor. PC-3 cells were isolated from a site of bone metastasis. These cells do not express androgen receptor. Both PC-3 and LNCaP C4-2 express higher levels of neuropilin-2 compared to LNCaP. The other known VEGF-C receptors VEGF-R3 and VEGF-R2 (data not shown) were detectable in significantly lower levels in LNCaP and LNCaP C4-2.
The presence of VEGF-C receptors on prostate cancer cells led us to look for a cancer cell specific function of VEGF-C. LNCaP C4-2 was selected initially, since it is an androgen responsive and androgen receptor-expressing prostate cancer cell line that recapitulates many features of hormone refractory human prostate cancers. This cell line also expresses high levels of the VEGF-C receptor Neuropilin-2, which we subsequently identified to be critical for VEGF-C function. We have observed an increase in apoptosis of LNCaP C4-2 cells when incubated for 5 hours with increasing concentrations of H2O2 (supplement figure 1). To investigate the survival-promoting role of VEGF-C, we first incubated serum starved LNCaP C4-2 cells with increasing concentrations of recombinant VEGF-C (9 hours) (R&D Systems Inc., Minneapolis, MN). After 4 hours of VEGF-C addition, cells were treated with a fixed dose of H2O2 (3mM) for the remaining 5 hours. Cell death was measured by the YO-PRO1/PI apoptosis assay using fluorescence microscopy. Our results (Figure 1B and 1C) suggested that prior addition of VEGF-C to these cells protects them from H2O2-induced cell death in a dose-dependent manner. In this respect it should be noted that LNCaP C4-2 and its syngenic parental line LNCaP express low level of endogenous VEGF-C, which showed moderate protection ability against a much lower (0.25mM) concentration of H2O2 (data not shown). Therefore, the endogenous VEGF-C present in LNCaP C4-2 cells in our experimental conditions (as described in Figure 1B and 1C) was not sufficient to protect cells against high levels of H2O2. We added exogenous VEGF-C for the following reason. It is known that in solid tumors VEGF-C is secreted by both tumor cells and inflammatory cells present in the tumor stroma (38). Therefore, the total level of VEGF-C should be higher than that secreted by the cancer cells alone. Equivalent concentrations of VEGF-C have been employed by other investigators (39), who demonstrated that these concentrations of VEGF-C are physiologically relevant.
In order to evaluate the signaling pathways important for VEGF-C mediated survival in prostate cancer cells under oxidative stress, we tested the regulation of AKT as a down-stream event. We observed a decrease in the phosphorylation status of AKT-1 (at Serine 473 and Threonine 308) in LNCaP C4-2 cells treated for 5 hours with increasing concentrations of H2O2 (Figures 2A, 2B and supplement figure 2). Prior incubation with VEGF-C abrogated the AKT-1 inactivation (Figure 2A) supporting the survival promoting function of VEGF-C as described in the previous result. No decrease in phospho-AKT-1 levels by H2O2 treatment were also observed when mature form of VEGF-C were stably overexpressed in LNCaP C4-2 cells (Figure 2B) (Relative expression levels of VEGF-C in the stable clones of C4-2 are described in the supplement Figure 3). These results emphasize the importance of VEGF-C in upregulating AKT-1 phosphorylation in prostate cancer cells during oxidative stress. It is important to note that VEGF-C mediated AKT-1 phosphorylation was observed only under oxidative stress conditions, as we detected no increase in phosphorylation of AKT-1 in the absence of H2O2 in LNCaP-C4-2 cells, either when incubated with recombinant VEGF-C or stably overexpressing mature VEGF-C (Figure 2A and 2B). The importance of this finding is confirmed by the fact that the AKT signaling cascade is considered a key determinant of tumor aggressiveness and an attractive target for therapeutic intervention (40). Also, cancer cells expressing constitutively active AKT are more resistant to chemotherapeutic drugs like paclitaxel and cisplatin than cancer cells expressing low levels of AKT (41).
Phosphorylation of FOXO-1 (at Threonine 24) and GSK3β (at Serine 9), both downstream targets of AKT-1, was also retained in VEGF-C treated cells (Figure 2A). FOXO-1, a known inducer of apoptosis loses its activity upon phosphorylation by AKT-1 (42). GSK3β is a mediator of c-Flip mediated apoptosis (43), and thus inactivation of GSK3β by phosphorylation suggests an anti-apoptotic function of VEGF-C.
Interestingly, VEGF-C could not restore the decrease in phosphorylation of S6 and 4EBP1, two downstream targets of AKT-mTOR complex 1 (mTORC-1) pathway in H2O2 treated prostate cancer cells (Figure 2C). Although, it is not clear why those proteins are not phosphorylated by VEGF-C under oxidative stress despite the presence of active AKT-1, an explanation might come from the observation that oxidative stress can block mTORC1 activity downstream of AKT-1 (44). Therefore, the inactivation of mTORC1 during severe stress may have biological significance. The major functions of mTORC1 are protein synthesis and cellular growth (45). It is possible that during severe stress cells prefer to shut off the energetically-expensive processes like protein synthesis and growth and enhance cellular processes that mediate cell survival. Again, one of the downstream targets of mTORC1 is S6 kinase, which apart from activating its substrate S6 is also involved in a negative feed-back loop to inactivate AKT-1 (46). Therefore, absence of mTORC-1 activity facilitates prolonged activation of AKT-1 during severe stress and thus provides a better survival advantage.
In order to understand the molecular pathway of VEGF-C mediated stress resistance in prostate cancer cells we studied the upstream events required for Serine 473 phosphorylation of AKT-1. Serine 473 phosphorylation of AKT was previously reported to be an excellent predictor of poor clinical outcome in prostate cancer (47). One of the important upstream candidates is the multi-protein complex mTOR complex 2 (mTORC2), which has shown to be necessary for prostate cancer development (46, 48). Accordingly, we tested the involvement of mTORC-2 in the VEGF-C-induced restoration of AKT-1 activation under ROS stress. We studied the association of mTOR and rictor, two important components of mTORC-2, in C4-2 cells treated with 3 mM H2O2 alone or in the presence of increasing doses of VEGF-C. A decrease in association of mTOR with rictor in prostate cancer cells was observed when treated with H2O2 alone (Figure 3A). Interestingly, prior incubation with VEGF-C restored the complex formation even in the presence of H2O2, suggesting a role of VEGF-C in maintaining mTORC-2 under stress (Figure 3A). The total expression level of mTOR and rictor were not influenced by the addition of H2O2 and/or VEGF-C (Figure 3A, lower two panels). Next, we knocked down the expression of rictor using specific siRNA in order to inactivate mTORC-2 in prostate cancer cells and monitored VEGF-C induced Serine 473 phosphorylation of AKT-1 under ROS stress. Recombinant VEGF-C failed to restore phospho-AKT-1 levels when rictor was knocked down (Figure 3B). These results together with the results in Figure 3A suggest the involvement of mTORC-2 in this pathway and rules out any significant contribution of other signaling pathways for VEGF-C mediated serine 473 phosphorylation of AKT-1 in prostate cancer cells during oxidative stress. Recent report also indicated the involvement of mTORC-2 for the progression of prostate cancer (48) underscoring the significance of our study.
In order to identify the VEGF-C receptor/s in this pathway, we showed interest in Neuropilin-2 because of its involvement in resisting metabolic stress (49). Furthermore, the Oncomine database demonstrated an increased expression of Neuropilin-2 in human metastatic prostate cancer tissues (23). Knocking down Neuropilin-2 in LNCaP C4-2 cells failed to rescue p-AKT-1 levels by VEGF-C when treated with 3 mM H2O2 (Figure 3C and 3D). Thus, our results demonstrate the involvement of Neuropilin-2 in VEGF-C mediated AKT-1 activation. Since Neuropilin-2 is a non-tyrosine kinase receptor and has a very short cytoplasmic tail, it is widely believed that it cooperates with other tyrosine kinase receptors to induce downstream signaling events (50). Currently the identity of the tyrosine kinase receptor, if any, in this process is unknown.
Finally, we also checked the VEGF-C- AKT-1 axis in other prostate cancer cells such as LNCaP and PC-3. Similar to LNCaP C4-2 cells, a dose dependent recovery of LNCaP cells from H2O2 induced apoptosis was observed with increasing concentrations of VEGF-C (Figure 4A right panel and Supplement figures 4 and 5). We also observed the phosphorylation of AKT-1 and its downstream target GSK-3β in LNCaP cells by the addition of recombinant VEGF-C during oxidative stress (Figure 4A left panel). Interestingly, unlike LNCaP C4-2 we only observed this restoration of phosphorylation at the lower concentrations of H2O2 (1 mM) (Figure 4A and Supplement Figure 6). The differences between LNCaP and C4-2 cells might be due to different neuropilin-2 expression levels in these two cell lines (Figure 1A). Neuropilin-2 expression is significantly higher in C4-2 cells and therefore should be more potent in resisting stress by VEGF-C.
Finally, we tested the stress-resistant function of VEGF-C in a highly metastatic prostate cancer cell line, PC-3, which not only express high level of neuropilin-2, but synthesize significant levels of endogenous VEGF-C. Decrease in phospho-AKT-1 levels in PC-3 cells by H2O2 were observed only at higher concentrations (7.5 and 10 mM) (Figure 4B left panel). As expected a rapid decrease in AKT-1 phosphorylation in PC-3 cells with increasing concentrations of H2O2 occurred (Figure 4B left panel) when endogenous expression of VEGF-C was knocked down by siRNA (10 nM). A significant increase in apoptosis in PC-3 cells was also observed when endogenous VEGF-C was knocked down (Figure 4B right panel, and supplement figure 7) along with the decrease in phosphorylation of FOXO-1 and GSK-3β (Figure 4C), confirming the requirement of VEGF-C in resisting ROS stress in PC-3 cells (Supplement figure 8 for the efficiency of VEGF-C knocking down in PC-3 by siRNA).
In conclusion, we have determined a novel function of VEGF-C, which promotes survival of prostate cancer cells under oxidative stress as schematically represented in Figure 4D. Involvement of Neuropilin-2 in this function of VEGF-C is intriguing because of its higher expression in metastatic prostate cancer. Since other cancers such as glioblastoma, osteosarcoma (our unpublished data) and several epithelial cancers also express higher levels of VEGF-C and Neuropilin-2, it is possible that the VEGF-C/Neuropilin-2/AKT-1 axis is involved in the recurrence of those cancers as well, a possibility that should be tested.
Research Scholar Grant from American Cancer Society (RSG-070944-01-CSM; K Datta); Career Development project in prostate SPORE grant (Mayo Clinic; 1 PSOCA91956-3; K Datta) and New Investigator award grant from US Army Medical Research and material Command (2B1636; K. Datta). NIH grants CA 121277 (D.J. Tindall), CA 91956 (D.J. Tindall), CA 125747 (D.J. Tindall), and the TJ Martell Foundation (FNDT Martell/#1–8; D.J. Tindall); and a SPORE Developmental project grant (M.H. Muders) and an Eagle’s grant (Eagles # 254, Eagle Foundation for Cancer Research; M.H. Muders).