Broad-spectrum G protein-coupled receptor antagonist, [D-Arg1,D-Trp5,7,9,Leu11]SP: a dual inhibitor of growth and angiogenesis in pancreatic cancer.

Substance P analogues, including [D-Arg(1),D-Trp(5,7,9),Leu(11)]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 Ca(2+) 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 Ca(2+) 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.

G protein-coupled receptor activation rapidly stimulates focal adhesion kinase phosphorylation at Ser-843. Mediation by Ca2+, calmodulin, and Ca2+/calmodulin-dependent kinase II.

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with the G protein-coupled receptor agonists bombesin, vasopressin, or bradykinin induced an extremely rapid (within 5 s) increase in FAK phosphorylation at Ser-843. The phosphorylation of this residue preceded FAK phosphorylation at Tyr-397, the major autophosphorylation site, and FAK phosphorylation at Ser-910. Treatment of intact cells with ionomycin stimulated a rapid increase in FAK phosphorylation at Ser-843, indicating that an increase in intracellular Ca2+ concentration ([Ca2+]i) is a potential pathway leading to FAK-Ser-843 phosphorylation. Indeed, treatment with agents that prevent an agonist-induced increase in [Ca2+]i (e.g. thapsigargin or BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), interfere with calmodulin function (e.g. trifluoperazine, W13, and W7), or block Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation (KN93) or expression (small interfering RNA) abrogated the rapid FAK phosphorylation at Ser-843 induced by bombesin, bradykinin, or vasopressin. Furthermore, activated CaMKII directly phosphorylated the recombinant COOH-terminal region of FAK at a residue equivalent to Ser-843. Thus, our results demonstrate that G protein-coupled receptor activation induces rapid FAK phosphorylation at Ser-843 through Ca2+, calmodulin, and CaMKII.

PDGF and FGF induce focal adhesion kinase (FAK) phosphorylation at Ser-910: dissociation from Tyr-397 phosphorylation and requirement for ERK activation.

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but very little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with platelet-derived growth factor (PDGF) promoted a striking increase in the phosphorylation of FAK at Ser-910, as revealed by site-specific antibodies that recognized the phosphorylated state of this residue. FAK phosphorylation at Ser-910 could be distinguished from that at Tyr-397 in terms of dose-response relationships and kinetics. Furthermore, the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 abrogated FAK phosphorylation at Tyr-397 but did not interfere with PDGF-induced FAK phosphorylation at Ser-910. Conversely, treatment with U0126, a potent inhibitor of MEK-mediated ERK activation, prevented FAK phosphorylation at Ser-910 induced by PDGF but did not interfere with PDGF-induced FAK phosphorylation at Tyr-397. These results were extended using growth factors that either stimulate, fibroblast growth factor (FGF), or do not stimulate (insulin) the ERK pathway activation in Swiss 3T3 cells. FGF but not insulin promoted a striking ERK-dependent phosphorylation of FAK at Ser-910. Our results indicate that FAK phosphorylation at Tyr-397 and FAK phosphorylation at Ser-910 are induced in response to PDGF stimulation through different signaling pathways, namely PI 3-kinase and ERK, respectively.

Differential coupling of the PAC1 SV1 splice variant on human colonic tumors to the activation of intracellular cAMP but not intracellular Ca2+ does not activate tumor proliferation.

PAC1 is a recently cloned and characterized heptahelical, G protein-coupled receptor with high affinity to PACAP-27 and PACAP-38 and is differentially coupled to activate intracellular Ca2+ and cAMP. PAC1 is expressed as four major splice variants, each possessing differential coupling to inositol phosphates and intracellular Ca2+. PAC1 has been shown previously to be expressed and regulate the growth and proliferation of nonsquamous cell lung cancer cells, as well as breast cancer cell lines. PAC1 is expressed on the HCT8 human colon cancer cell line and is coupled to the activation of both intracellular cAMP and Ca2+ with consequent stimulation of growth. In the current study, we contrast the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the HCT8 colon cancer cell lines to the HCT116 and FET cell lines wherein PAC1 is expressed as the SV1 or HIP splice variant and is coupled to the activation only of cAMP but not of intracellular Ca2+. These data indicate that human colon tumor cells express PAC1 and are differentially coupled to intracellular signal transduction molecules. The ability to activate both cAMP and Ca2+ appears to be a prerequisite for activation of tumor proliferation, indicating a potentially important factor in how PACAP potentiates the growth of certain tumors.

The role of transforming growth factor alpha in determining growth factor independence.

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.