Substance P analogues, including [D-Arg1,D-Trp5,7,9,Leu11]SP (SPA) are broad-spectrum G protein–coupled receptor (GPCR) antagonists that have potential antitumorigenic activities, although the mechanism(s) are not completely understood. Here, we examined the effects of SPA in ductal pancreatic cancers that express multiple GPCRs for mitogenic agonists and also produce proangiogenic chemokines. Using HPAF-II, a well-differentiated pancreatic cancer cell line as our model system, we showed that SPA inhibited multiple neuropeptide-induced Ca2+ mobilization, DNA synthesis, and anchorage-independent growth in vitro. SPA also significantly attenuated the growth of HPAF-II tumor xenografts in nude mice beyond the treatment period. Interestingly, SPA markedly increased apoptosis but moderately decreased proliferation marker, Ki-67 in the tumor xenografts implying additional mechanism(s) for the significant growth inhibitory effect observed in vivo. HPAF-II cells express ELR+ CXC chemokines, including IL-8/CXCL8, which bind to CXCR2 (a member of GPCR superfamily) and promote angiogenesis in multiple cancers, including pancreatic cancer. SPA inhibited CXCR2-mediated Ca2+ mobilization and blocked specifically IL-8/CXCL8-induced angiogenesis in rat corneal micropocket assay in vivo. A salient feature of the results presented here is that SPA markedly reduced tumor-associated angiogenesis in the HPAF-II xenografts in vivo. Our results show that SPA, a broad-spectrum GPCR antagonist attenuates tumor growth in pancreatic cancer via a dual mechanism involving both the antiproliferative and antiangiogenic properties. We conclude that this novel dual-inhibitory property of SPA could be of significant therapeutic value in pancreatic cancer, when used in combination with other antiproliferative and/or antiangiogenic agents.
Pancreatic ductal adenocarcinoma or pancreatic cancer is the most fatal gastrointestinal malignancy, with only 3% to 5% overall 5-year survival rate (1). Pancreatic cancer is mostly refractory to current therapeutic regimens, rendering it nearly 100% lethal, and making it now the fourth leading cause of cancer death in both men and women (1). Thus, novel therapeutic strategies are urgently required, and these will most likely arise from a better understanding of the factors and signaling pathways that stimulate the proliferation of ductal pancreatic cancer cells (2).
Neuropeptide agonists and their cognate G protein–coupled receptors (GPCR) are increasingly implicated as autocrine/paracrine growth factors for multiple solid tumors including small cell lung cancer (SCLC), colon, breast, prostate, and pancreas (3, 4). We showed that pancreatic cancer cell lines express multiple functional GPCRs using Ca2+ mobilization assay as indicator of productive ligand-receptor interactions (5). A variety of neuropeptides including neurotensin, bradykinin, and vasopressin stimulated DNA synthesis in multiple pancreatic cancer cell lines (5–7).5
Substance P analogues, including [D-Arg1, D-Phe5, D-Trp7,9, Leu11]SP and [Arg6, D-Trp7,9, MePhe8]SP (SPG, refs. 6–11) block the biological effects of a broad range of GPCR agonists structurally unrelated to substance P in multiple cell types (11, 12). These broad-spectrum GPCR antagonists also inhibit the proliferation of SCLC cell lines in liquid culture, in soft agar, and as xenografts in nude mice (11, 12). Thus, SPG has recently completed a phase I clinical trial with minimal toxicity (facial flushing) and successfully blocked substance P–induced vasodilatory effects in vivo with no dose-limiting toxicity (13). Recently, a more potent GPCR antagonist, [D-Arg1, D-Trp5,7,9, Leu11]SP or substance P antagonist (SPA), has been identified that also inhibited SCLC cell proliferation both in vitro and in vivo (14). However, it is not known whether SPA can block GPCR-mediated angiogenesis in tumors.
Given the fact that pancreatic cancer cells, including HPAF-II ductal adenocarcinoma cells express multiple GPCRs that mediate mitogenic signaling and produce proangiogenic ELR+ CXC chemokines, including IL-8/CXCL8 (10, 15), we examined the effects of the potent broad-spectrum GPCR antagonist, SPA, in these cells growing in vitro and in vivo. We show that SPA blocked multiple neuropeptide-induced [Ca2+]i mobilization, decreased DNA synthesis, and anchorage-independent growth of HPAF-II cells in vitro. SPA also significantly attenuated the growth of established HPAF-II tumor xenografts in vivo beyond the treatment period and markedly increased apoptosis. Interestingly, SPA specifically and strikingly blocked IL-8/CXCL8-induced angiogenesis in the rat corneal micropocket assay and also significantly reduced tumor-associated angiogenesis of the HPAF-II xenografts in vivo. We conclude that SPA inhibits tumor growth via a dual mechanism involving both antimitogenic effects and a previously unrecognized antiangiogenic activity that could be of significant therapeutic values in pancreatic cancer.
Materials and Methods
Cell culture. HPAF-II, obtained from American Type Culture Collection (Manassas, VA), is a well-differentiated line established from human ductal pancreatic adenocarcinoma. HPAF-II cells were grown in RPMI 1640 (Sigma, St. Louis, MO) with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO2. HEK-293 cells stably transfected with CXCR2 were grown in G-418 containing DMEM (Sigma) with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2.
Measurement of intracellular calcium. Intracellular Ca2+ concentration ([Ca2+]i) was measured with the fluorescent indicator fura-2 as previously described (5). Agonists and/or antagonists were added at various time points during recording.
[3H]-Thymidine incorporation. DNA synthesis was measured using [3H]-thymidine incorporation assay as previously described (6).
Anchorage-independent cell growth. Cells (2 × 104) in either RPMI 1640 + 1% FBS, or RPMI 1640 + 10% FBS (positive control) were plated on tissue culture 12-well plates coated with polyhydroxyethylmethacrylate [poly-(HEMA)]. The growth of these cells in suspension was measured as previously described (8).
Xenografts. The HPAF-II xenograft was derived by implantation of 2 × 107 cells of the HPAF-II cell line into the right flanks of the male nu/nu mice. Histologic analysis confirmed the pathology of these xenografts.
Animals. Male nu/nu mice were maintained in specific pathogen-free facility at University of California at Los Angeles (UCLA). The UCLA Chancellor's Animal Research Committee approved all the animal experiments.
Antitumor testing. The animals were randomized into control and treated groups (12 mice per group) and were given punched ear tags to allow identification. Treatment was initiated when the tumors reached a mean diameter of 6 mm (initial experiment) and subsequently when the tumors reached a mean diameter of 2 mm, and the 1st day of treatment in both cases was designated as day 0. Tumor volume (V) was estimated as V = 2/3 πr3, where r is the mean of the three dimensions (length, width, and depth). For injection into animals, [D-Arg1,D-Trp5,7,9,Leu11]SP (SPA) was dissolved in sterile water and was given once-daily peritumorally at 35 μg per g per day (50 μL/mouse) for 10 days.
Ki-67 immunohistochemistry. Cryostat sections (5 μm) were fixed in 95% ethanol, and stained with anti-Ki-67 (rabbit monoclonal clone SP6, NeoMarkers, Fremont, CA) antibody as previously described (16).
Microvessel density. Cryostat sections (5 μm) were fixed in acetone and stained with a rat anti-mouse CD31 monoclonal antibody (PharMingen, San Diego, CA) as previously described (17). Areas of greatest vessel density were then examined under higher magnification (100×) and counted. Any distinct area of positive staining for CD31 was counted as a single vessel. Results were expressed as the mean number of vessels ± SE per high-power field (HPF or 100×). A total of 20 HPFs were examined and counted from three tumors of each of the treatment groups.
Human cytokine expression array assay. The human cytokine array 5.1 was purchased from Ray Biotech (Norcross, GA) and used following the manufacturer's instructions (18).
IL-8/CXCL8 ELISA. Antigenic IL-8 was quantitated using a modification of a double-ligand ELISA method as previously described (19).
Rat corneal micropocket assay. In vivo angiogenic activity of the tumors was assayed in the avascular cornea of Long Evans rat eyes, as previously described (20).
In situ terminal deoxynucleotidyl transferase–mediated nick end labeling assay. Cryostat sections (5 μm) were fixed in 4% paraformaldehyde (in PBS, pH 7.4), and in situ terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assay (Roche Diagnostics, Germany) was done as per the manufacturer's instructions described previously (21).
Materials. [γ-32P] ATP (370 MBq/mL) was obtained from Amersham, plc. (Buckinghamshire, United Kingdom). Neurotensin, angiotensin-II, bombesin, bradykinin, vasopressin, cholecystokinin, RPMI 1640, and poly-(HEMA) were purchased from Sigma. [D-Arg1, D-Trp5,7,9, Leu11]SP (SPA) was obtained from Bachem, Inc. (Torrance, CA). All other reagents were of the purest grade available.
[D-Arg1,D-Trp5,7,9,Leu11]SP prevents multiple G protein–coupled receptor agonist-induced increase in [Ca2+]i, DNA synthesis, and anchorage-independent growth in HPAF-II cells. HPAF-II cells have been extensively used as a model system to study the effects of growth factors on the biological behavior of human pancreatic cancer cells (22–24). In addition, the histology sections of the HPAF-II tumors developed either in orthotopic or xenograft nude mice models closely resemble features of human pancreatic ductal adenocarcinoma (25). Thus, we used HPAF-II cells as our model system to study the effects of a broad-spectrum GPCR antagonist, [D-Arg1,D-Trp5,7,9,Leu11]SP (SPA), both in vitro and in vivo.
One of the earliest events induced by many GPCR agonists, including neurotensin, bombesin/gastrin releasing peptide, and vasopressin is a rapid phospholipase Cβ-mediated hydrolysis of phosphatidyl inositol-4,5-bisphosphate to produce the second messenger inositol-1,4,5-trisphosphate, which promotes mobilization of Ca2+ from intracellular stores (5). In agreement with our previous results (5), addition of multiple GPCR agonists including angiotensin, neurotensin, bombesin/gastrin releasing peptide, bradykinin, cholecystokinin, and vasopressin induced rapid [Ca2+]i in HPAF-II cells (Fig. 1D). This substantiates that HPAF-II cells express functional GPCRs for multiple agonists. Representative tracings shown in Fig. 1A , B, and C, show that pretreatment with SPA potently blocked the transient increase in [Ca2+]i induced by neurotensin, bombesin, and vasopressin in HPAF-II cells. These results show that SPA, which is structurally unrelated to neurotensin, bombesin, and vasopressin can act as a broad-spectrum GPCR antagonist in the human pancreatic cancer HPAF-II cells.
Pancreatic cancer cells, including HPAF-II produce mitogenic GPCR ligands, which can promote proliferation in an autocrine/paracrine manner (26). As a first step to determine the mechanisms underlying the direct antiproliferative properties of SPA, we examined its effects on the incorporation of [3H]-thymidine into DNA of HPAF-II cells. Confluent cultures of HPAF-II cells grown in medium containing 10% FBS were washed and transferred to fresh medium containing 1% FBS. To start the experiment, SPA at defined concentrations or solvent was added to parallel cultures for 16 hours and pulse labeled for 6 hours with [3H]-thymidine. As shown in Fig. 1E, treatment with increasing concentrations of SPA reduced the DNA synthesis of HPAF-II cells in a concentration-dependent fashion.
Next, we investigated whether SPA could block the proliferation of HPAF-II cells growing in an anchorage-independent fashion, a hallmark of transformed cells. To test this possibility, single cell suspensions of HPAF-II cells were plated in medium containing 1% FBS and SPA or solvent on culture dishes coated with poly-(HEMA), which prevents adhesion of the cells to the substratum. As illustrated in Fig. 1F, addition of increasing doses of SPA significantly inhibited (by 50%) HPAF-II cell numbers after 14 days of incubation. Our results show that SPA directly attenuates the proliferation of HPAF-II cells in vitro.
Effect of [D-Arg1,D-Trp5,7,9,Leu11]SP on growth of HPAF-II tumor xenografts in nude mice. Based on the antiproliferative effect of [D-Arg1,D-Trp5,7,9,Leu11]SP (SPA) in vitro, we next examined whether SPA could inhibit pancreatic cancer growth using HPAF-II tumor xenografts in nude mice. We used two distinct models to analyze the growth-inhibitory effect of SPA in vivo. Initially, we used an established tumor xenograft model to emulate the clinical scenario usually observed in pancreatic cancer. Specifically, we analyzed the effect of SPA in HPAF-II tumor xenografts that grew to an approximate volume of 150 mm3, which were generated by implanting 2 × 107 cells in the right flanks of the animals. Figure 2A shows that peritumoral injection of SPA at 35 μg per g per day for 10 days in the established HPAF-II xenograft produced a significant inhibition of tumor growth (P < 0.05) after 15, 20, and 25 days of initiating the 10-day treatment protocol. The representative H&E-stained sections of the xenograft tumors treated with vehicle show well-differentiated dysplastic ductal structures with characteristic arborization, network formation, dilated cystic spaces, and minimal to moderate desmoplastic changes (Fig. 2B,, top). In contrast, a representative section of the treated HPAF-II tumors show well-differentiated dysplastic ductal structures in the periphery but prominent necrotic areas in the center (Fig. 2B , bottom).
Furthermore, we examined the effect of SPA in the xenograft model starting 3 days after the implantation of HPAF-II cells with approximate initial tumor volume of 50 mm3. This near-concurrent peritumoral administration of SPA simulates an in vivo model for tumors initiating metastatic processes. As shown in Fig. 2C, peritumoral injections of SPA virtually suppressed the growth of HPAF-II xenograft during the treatment period. Inhibition of tumor growth was maintained for at least 18 days after initiation of the SPA treatment (Fig. 2C , inset). Our results indicate that administration of SPA significantly inhibits the growth of pancreatic cancer cells xenografted in nude mice.
Effect of [D-Arg1,D-Trp5,7,9,Leu11]SP on Ki-67 expression and in situ apoptosis of HPAF-II tumor xenografts. The results presented in Fig. 2 prompted us to investigate the mechanisms of the growth-inhibitory effects of [D-Arg1,D-Trp5,7,9,Leu11]SP (SPA) in vivo by analyzing the Ki-67 expression in the HPAF-II tumor xenografts. The expression of Ki-67 correlates well with other variables of cell proliferation including, thymidine labeling index, S-phase fraction, and mitotic count (27). Figure 3A illustrates representative tumor sections divided into central and peripheral zones labeled with Ki-67 SP-6 antibody (nuclear brown dots). Treatment with SPA significantly decreased Ki-67 labeling in the nonnecrotic tumor center (36%) compared with the periphery of the tumor (13%). Overall, SPA treatment only had a modest effect (22%) in reducing Ki-67 expression in HPAF-II tumor xenografts (Fig. 3A , inset).
Having documented that SPA decreased proliferation, we next examined whether SPA increased apoptosis in these tumor tissues. DNA degradation is considered a key event in apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments (28). We detected DNA strand breaks in situ by TUNEL assay on frozen sections of the HPAF-II xenografts. The fluorescein-labeled tissue was next mounted in a solution containing 4′,6′-diamino-2-phenylindole, which stains the nuclei. As shown in Fig. 3B, SPA treatment markedly increased apoptosis (by 43%) compared with vehicle control in the tumor xenografts. Interestingly, exposure to SPA (1-20 μmol/L) for 24 hours did not induce apoptosis in cultures of HPAF-II cells (data not shown). Thus far, our results suggest that SPA had a moderate growth-inhibitory effect in vivo. However, this does not explain the marked increase in apoptosis of the SPA-treated HPAF-II tumor xenografts.
Effect of [D-Arg1,D-Trp5,7,9,Leu11]SP on IL-8/CXCL8 production in HPAF-II cells, IL-8/CXCL8-induced increase in [Ca2+]i in HEK-293-CXCR2+ cells, IL-8/CXCL8-induced angiogenesis and tumor-associated angiogenesis in vivo. To further explain the significant increase in apoptosis and central necrosis observed in [D-Arg1,D-Trp5,7,9,Leu11]SP (SPA) treated tumors, we investigated whether SPA could also block angiogenesis in the HPAF-II xenografts. Inhibition of angiogenesis could indirectly promote apoptosis without significantly affecting tumor cell proliferation (29). ELR+ CXC chemokines share a common chemokine receptor, CXCR2 and strongly promote tumorigenesis in human NSCLC xenograft models in severe combined immunodeficient mice (9). Thus, we hypothesized that SPA, a broad-spectrum GPCR antagonist, could block the proangiogenic effects of ELR+ CXC chemokines during tumorigenesis and correspondingly increased apoptosis of the tumor xenografts.
Initially, using a human cytokine microarray assay, we screened the expression profile of cytokines from serum-starved confluent HPAF-II cells. The microarray membrane was immobilized with capture antibodies against 79 different cytokines (Supplementary Fig. S5). The membranes were hybridized with 1 mL of supernatant from SPA-treated (20 μmol/L, for 16 hours), or vehicle-treated (control) HPAF-II cells. The corresponding proteins were detected by a mixture of detection antibodies and visualized by an enhanced chemiluminescence system. Although HPAF-II cells produce multiple ELR+ CXC chemokines, we used IL-8/CXCL8 as the representative proangiogenic chemokine for the current study. As shown in Fig. 4A,, left (circled), IL-8/CXCL8 expression was detected in control HPAF-II cells and was not diminished by SPA treatment. Subsequently, we confirmed by ELISA that HPAF-II cells produce IL-8/CXCL8 and increasing concentrations of SPA did not block the IL-8/CXCL8 production (Fig. 4A , right). Next, we observed that HPAF-II cells do not express CXCR2, both at the mRNA and protein levels (data not shown). Thus, our results show that SPA did not block the IL-8/CXCL8 production by the HPAF-II cells.
Previous studies have reported that a related broad-spectrum GPCR antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]SP, binds to IL-8 receptors (CXCR1 and CXCR2) on human neutrophils (30), but the effect of SPA on CXCR2-mediated angiogenesis has not been explored in any system. Using IL-8/CXCL8 and CXCR2 as representative ELR+ CXC chemokine and its corresponding receptor, respectively, we examined whether SPA could block CXCR2-mediated rapid signaling events in HEK-293 cells stably transfected with CXCR2. As shown in Fig. 4B, SPA completely abrogated the rapid increase in [Ca2+]i induced by IL-8/CXCL8 in this model system. This strongly suggests that the inhibitory effect of SPA on IL-8/CXCL8-mediated downstream signaling events is at the level of CXCR2, a known GPCR. Next, we investigated whether SPA could block IL-8/CXCL8 induced angiogenesis in the avascular cornea of Long Evans rat eyes. We also tested the effect of SPA on angiogenesis induced by basic fibroblast growth factor (bFGF), which acts through a tyrosine kinase receptor. Previous data showed that SPA does not interfere with the biological effects of ligands of tyrosine kinase receptors (31). Rat corneas were anesthetized and subsequently sterile Hydron pellets containing recombinant IL-8/CXCL8 (80 ng per pellet), or recombinant bFGF (50 ng per pellet) combined with vehicle control or SPA (10 μmol/L) were implanted into an intracorneal pocket (1-2 mm from the limbus). Six days after implantation, the animals were perfused with colloidal carbon, the corneas were harvested and photographed. As shown in Fig. 4C, both IL-8/CXCL8 and bFGF potently increased neovascularization responses towards the implant. Interestingly, SPA markedly blocked IL-8/CXCL8-induced angiogenesis (four of six corneas) but not bFGF-induced angiogenesis (zero of six corneas) in the rat corneas (Supplementary Table S1). Thus, our results show that SPA significantly blocked IL-8/CXCL8-induced angiogenesis in vivo and the inhibitory action is at the level of the receptor, specific to the GPCR family.
Having established that SPA could block ELR+ CXC chemokine-induced angiogenesis, we next examined whether SPA reduced microvessel formation in the tumor xenografts. It is well established that CD31 or platelet/endothelial cell adhesion molecule-1 (PECAM-1) is an adhesion molecule expressed on mature vascular endothelial cells and has been extensively used as a specific marker for microvessel formation (to calculate microvessel density) in the tumor sections (25). Thus, to detect tumor-associated neovascularization, we did immunohistochemistry on frozen sections from the HPAF-II xenografts with rat anti-mouse CD 31/PECAM-1 antibody. As shown in Fig. 4D, SPA treatment significantly reduced (by 44%) CD31+ vessels/mm2 in HPAF-II tumor xenografts. Taken together, our results suggest that SPA markedly reduced tumor-associated angiogenesis in the xenografts by acting on the host vascular endothelial cells. This is a novel property of broad-spectrum GPCR antagonist, SPA.
GPCRs that mediate agonist-induced signal transduction and cancer cell proliferation are attracting attention because they may provide potential targets for novel therapeutic interventions. HPAF-II pancreatic cancer cells, our model system in this study, express GPCRs for multiple mitogenic agonists and also produce proangiogenic ELR+ CXC chemokines, including IL-8/CXCL8. Given the fact that GPCR agonists function as autocrine/paracrine growth factors for multiple cancers, including pancreatic cancer, we investigated whether the broad-spectrum GPCR antagonist, [D-Arg1,D-Trp5,7,9,Leu11]SP (SPA) could block growth of HPAF-II cells both in vitro and in vivo.
The results presented in this paper illustrate that SPA is a broad-spectrum GPCR antagonist that significantly reduced DNA synthesis and growth in suspension of the HPAF-II cells in vitro. SPA also attenuated growth of established HPAF-II tumor xenografts beyond the treatment period and reduced Ki-67 expression in vivo. However, SPA markedly increased apoptosis in vivo. In addition, in contrast to studies showing that substance P derivatives can promote apoptosis of SCLC cells in culture (32), we did not observe any direct proapoptotic effect of SPA on HPAF-II cells in vitro. Interestingly, of the two HPAF-II xenograft models, we observed a prominent effect of SPA on the established tumor, which is quite akin to the clinical scenario in pancreatic cancer. In this model, SPA significantly attenuated growth beyond the treatment period, which could not be explained only by its antiproliferative property. Thus far, our results show that SPA not only has direct growth-inhibitory effects in vitro and in vivo but also has additional mechanism(s) to promote significant apoptosis and central necrosis observed in vivo. One of the mechanism could be inhibition of tumor-associated angiogenesis, as it was previously suggested that an established tumor predominantly depends on it for further growth (29).
A salient feature of this paper is that SPA significantly decreased tumor-associated angiogenesis and correspondingly increased apoptosis of HPAF-II xenografts in vivo. This is a novel finding for the group of broad-spectrum GPCR antagonists, including [Arg6, D-Trp7,9, MePhe8]SP or SPG (6–11), which is entering phase II clinical trial for SCLC. Our results showed that SPA specifically blocked IL-8/CXCL8 (member of ELR+ CXC chemokines) and not bFGF-mediated corneal neovascularization in vivo. HPAF-II cells produce ELR+ CXC chemokines, including IL-8/CXCL8 but do not express their corresponding receptor, CXCR2 (also a member of GPCR superfamily). CXCR2, predominantly expressed on the endothelial cells, is an important mediator of angiogenesis in multiple cancers. Here, we showed that SPA blocked CXCR2-mediated intracellular Ca2+ mobilization. Thus, SPA blocked angiogenesis in HPAF-II tumor xenografts by inhibiting CXCR2-mediated signaling events in the host vascular endothelial cells. Taken together, this antiangiogenic property of SPA along with its growth-inhibitory effects could explain the pronounced and sustained growth attenuation observed in treated HPAF-II tumor xenografts.
In conclusion, our results raise the attractive possibility that treatment with SPA, a potent broad-spectrum GPCR antagonist, sustains growth inhibition in vivo by two different mechanisms: direct inhibition of cancer cell proliferation and by a previously unrecognized interference of the angiogenic properties of the HPAF-II tumor xenografts. The results provide a basis for novel noncytotoxic therapeutic strategies for the treatment of pancreatic cancer, a devastating disease with limited survival options.
Note: S. Guha and G. Eibl contributed equally to the work.
Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Grant support: Ronald S. Hirshberg Memorial Foundation for Pancreatic Cancer Research; NIH grant DK55003; and National Cancer Institute grant P50CA90388; Department of Medicine, David Geffen School of Medicine at UCLA specialty training and advanced research fellowship (S. Guha); and AGA Mentors' research scholar award (S. Guha).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the members of the Rozengurt laboratory for many valuable discussions and Rodney Miller, M.D. (ProPath Laboratory, Inc., Dallas, TX) for valuable help with Ki-67 immunohistochemical staining.