Peptide receptors as cancer drug targets

Abstract

Neuropeptides function as neuromodulators in the brain whereby they are released in a paracrine manner and activate G protein-coupled receptors (GPCRs) in adjacent cells. Because neuropeptides are made in and secreted from cancer cells, then bind to cell surface receptors, they function in an autocrine manner. Bombesin (BB)-like peptides synthesized by neuroendocrine tumor small cell lung cancer (SCLC) bind to BB receptors (BBR) causing phosphatidylinositol turnover and phosphorylation of extracellular-signal-regulated kinase (ERK). Phosphorylated ERK enters the nucleus and alters gene expression of SCLC cells, stimulating growth. Vasoactive intestinal peptide (VIP) addition to SCLC cells increases their release rate of BB-like peptides via activation of VIP receptors (VIPR), leading to activation of adenylyl cyclase and subsequent elevation of cAMP. Protein kinase A is then stimulated, leading to phosphorylation of CREB, which alters gene expression and stimulates proliferation. The growth of SCLC is inhibited by BBR and VIPR antagonists. This review will focus on how GPCRs for VIP and BB are molecular targets for early detection and treatment of cancer.

INTRODUCTION

Neuropeptides are biologically active in the central nervous system and periphery (1). Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide that enhances survival of spinal cord neurons in culture (2); VIP is neuroprotective because it causes secretion of cytokines and growth factors from astroglia (3). VIP and pituitary adenylate cyclase activating polypeptide (PACAP) stimulate the growth of lung cancer cells (4,5). PACAP-27 has 67% sequence homology with VIP; however, VIP binds with high affinity to VPAC1 and VPAC2, whereas PACAP binds with high affinity to VPAC1, VPAC2, and PAC1 (6). These receptors interact with G_s, increasing adenylyl cyclase activity and resulting in elevated cAMP. cAMP increases protein kinase (PK) A activity resulting in phosphorylation of CREB (7), followed by altered gene expression and increased proliferation. The growth of lung cancer cells is inhibited by VIPhyb, which blocks VPAC1 and VPAC2, and by PACAP (6–38), which antagonizes PAC1 (8).

Addition of VIP to lung cancer cells increases the secretion rate of bombesin (BB)-like peptides from the cells (9). BB and the structurally related gastin releasing peptide (GRP) bind with high affinity to BB₂R, causing phosphatidyl inositol (PI) turnover (10). Neuromedin B (NMB), which has homology with the C-terminal of BB, binds with high affinity to the BB₁R. Addition of GRP or NMB to lung cancer cells results in increased tyrosine phosphorylation of ERK (11); phosphorylated ERK then enters the nucleus and alters expression of the genes FOS and JUN, leading to growth proliferation (12). In contrast, BB₁R or BB₂R antagonists such as PD168368 or BW2258U89 inhibit lung cancer growth (13,14).

In this review I will focus on how G protein-coupled receptors (GPCRs) for VIP and BB are targets for the early detection and treatment of cancer.

VIP/PACAP

VIP is derived from a 170 amino acid precursor protein (7). VPAC1, which is present in high densities (~100,000 molecules/cell) in lung cancer cells, binds VIP and PACAP with high affinity, and VIPhyb with moderate affinity (Table I). In contrast, PACAP-27 and PACAP-38 are derived from a different 176 amino acid precursor protein (6). The PACAP gene (ADCYAP1) is hypermethylated in cervical cancer [15]. PACAP-27 and PACAP-38 bind with high affinity to PAC1, VPAC1, and VPAC2, however PACAP (6–38) is an antagonist for PAC1 (16). Selective agonists for VPAC1, VPAC2, and PAC1 are (Lys¹⁵, Arg¹⁶, Leu¹⁷) VIP¹⁻⁷GRF⁸⁻²⁷, Ro25–1553, and (Iaa¹, Ala^16,17, D-Lys³⁸ (IAAD)) PACAP-38, respectively (17). Recently, small molecule PAC1 antagonists PA-8 and PA-9 were discovered (18). It remains to be determined if PA-8 or PA-9 inhibits the proliferation of cancer cells.

Table I.

Binding of VIP/PACAP-like peptides

Addition	IC50 nM
	125I-VIP binding	125I-PACAP-27 binding
PACAP-27	2 ± 0.4	3 ± 0.5
VIP	2 ± 0.5	>1000
PACAP (6–38)	> 1000	30 ± 4
(SN)VIPhyb	30 ± 4	> 1000
(Lys15, Arg16, Leu17)VIP(1–7)GRF(8–27)	10 ± 2	>1000
Ro25–1553	> 1000	> 1000
(IAAD)-PACAP-38	18 ± 3	0.3 ± 0.1
(ANK)VIP-L2-CPT	150 ± 20	>1000
VIP-LALA-E	500 ± 50	>1000

The IC₅₀ (nM) to inhibit specific ¹²⁵I-PACAP-27- or ¹²⁵I-VIP binding to NCI-H1299 cells is indicated. The mean value ± S.D. of 3 experiments each repeated in quadruplicate is indicated. The structures of PACAP-27 and VIP, with homologies relative to PACAP-27, underlined are:

PACAP-27: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH₂.

VIP: His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Glu-Ser-Ile-Leu-Asn-NH₂.

VPAC1, VPAC2, and PAC1 are type II (secretin) GPCRs that interact with stimulatory guanine nucleotide binding proteins (G_s). VPAC1, which contains 457 amino acids, is present in high densities in bladder, breast, colon, liver, lung, pancreatic, prostate, thyroid, and uterus cancers (19, 20). VIP-Leu-Ala-Leu-Ala-ellipticine (LALA-E) binds to VPAC1 with moderate affinity and is internalized (21). LALA-E is metabolized in lysozomes and cytotoxic E is released into the cells, where it inhibits proliferation and topoisomerase II activity. Because the N-terminus of VIP is essential for biological activity, the C-terminus can be modified with various drugs. (Ala^{2,8,9,11,19,24,25.27}, Nle¹⁷, Lys¹⁸ (ANK))VIP coupled to camptothecin (CPT) has been shown to inhibit lung cancer proliferation and topoisomerase I activity (22), thus VPAC1 is a target that can be used to deliver cytotoxic agents into cancer cells.

VPAC2, which contains 438 amino acids, is present in moderate densities in gastric, pancreatic adenocarcinomas, gastric leiomyomas, and thyroid cancers, and in sarcomas. PAC1, which contains 468 amino acids, is present in moderate densities in brain, breast, colon, lung, neuroendocrine, pancreatic, pituitary, and prostate cancers. There is approximately 50% sequence homology between VPAC1, VPAC2, and PAC1; however, PAC1 has splice variants (SV): addition of a 28 amino acid segment to intracellular loop (IL) 3 of PAC1null results in PAC1hip; addition of a different 28 amino acid segment results in PAC1hop; addition of both segments to PAC1null results in PAC1hiphop. PAC1 can interact with G_q, stimulating PI turnover. The order of potency to cause PI turnover is PAC1hop > PAC1null = PAC1hiphop > PAC1hip (23). The gene PAC1 is composed of 18 exons, and deletions at its N-terminus have been observed in neuroblastoma cells (24). Deletions of exons 5, 6, or 4–6 result in deletions of 7 amino acids, 21 amino acids (PAC1-short, PAC1s), or 57 amino acids (PAC1-very short; PAC1vs), respectively. PAC1s, but not PAC1vs, binds to PACAP-38 with high affinity and when cAMP is elevated (25). Deletion of 53 amino acids from the C-terminus of PAC1 impairs signal transduction and receptor desensitization (26). Site-directed mutagenesis of PAC1 has indicated that Arg⁴¹⁶ and Ser⁴¹⁷ are essential for its inactivation. These results indicate that PAC1 binding and signal transduction can be altered by deletions or mutations of PAC1.

Peptide GPCRs are present in both neuroendocrine (SCLC) and epithelial cancers (non-SCLC). GPCRs are present in NSCLC cells, as are receptor tyrosine kinases (RTK) such as epidermal growth factor receptor (EGFR) and HER2. Addition of 100 nM PACAP-27 or PACAP-38 to NSCLC cells significantly increases tyrosine phosphorylation of EGFR and HER2 within 1 minute (27). EGFR and HER2 transactivation are inhibited upon addition of PACAP (6–38), gefitinib (EGFR tyrosine kinase inhibitor (TKI)), PP2 (Src inhibitor), TGF-α antibody, or GM6001 (matrix metalloprotease inhibitor (MMP) inhibitor) (Fig. 1). The activation of MMP may metabolize pro-TGF-α into TGF-α, which binds to and activates EGFR (28). In contrast, there is no known ligand for HER2; however, HER2 can form heterodimers with EGFR (activated EGFR usually forms homodimers with itself). The Ras–Raf–MEK–ERK proliferation pathway is then activated, resulting in phosphorylation of ERK; pERK then enter the nucleus and alters gene expression leading to increased proliferation. The PI3K pathway is also activated, leading to increased cellular survival. Addition of VIP to breast cancer cells increases tyrosine phosphorylation of EGFR and HER2 (29).

Fig. 1.

Effect of GPCRs on RTK transactivation. GPCRs for BB and PACAP couple to Gq11 and causes metabolism of PIP₂ to DAG (activates PKC) and IP₃ (elevates cytosolic Ca²⁺). GPCRs for VIP and PACAP interact with G_s activating adenylyl cyclase and increasing cAMP. cAMP activates PKA leading to CREB phosphorylation and altered gene expression. GPCRs activate Src and MMP, resulting in the production of EGFR ligands such as TGF-α. When TGF-α binds to the EGFR, tyrosine kinase activity is increased leading to phosphorylated EGFR homodimers or EGFR–HER2 heterodimers. RTK activates the Ras–Raf–MEK–ERK pathway, leading to increased cellular proliferation. RTK also activates the PI3K–PKD–Akt–mTor pathway, leading to increased cellular survival. Phosphorylated EGFR–HER2 is dephosphorylated by protein tyrosine phosphatase (PTP), which is impaired by ROS.

A surprising finding is that EGFR transactivation requires reactive oxygen species (ROS). The increase in EGFR tyrosine phosphorylation caused by PACAP is inhibited by N-acetyl cysteine (NAC is an antioxidant), Tiron (superoxide scavenger), or diphenylene iodonium (DPI). DPI inhibits the enzymes Nox and Duox that synthesize ROS. ROS oxidize Cys⁷⁹⁷ of the EGFR, increasing tyrosine kinase activity and decreasing enzymatic activity of protein tyrosine phosphatases (PTP), impairing phosphotyrosine degradation (30). Addition of PACAP to NSCLC increases ROS; this can be impaired by NAC, Tiron, or DPI. Also, DPI inhibits the proliferation of NSCLC cells.

A goal is to image cancer early when it is more amenable to treatment. VIP and PACAP can be radiolabeled to image cancer tumors. Initially, ¹³¹I-VIP was used to image tumors in colon cancer patients (31). Subsequently ⁶⁴Cu-TP3982- or ¹⁸F(Arg^15,21)VIP was used to image mammary tumors in mice (32,33). ^99mTc-TP3982VIP has been used imaged breast tumors in five patients (34). VPAC1 has a higher density in mammary tumors relative to adjacent normal tissue (35). Because VIPhybrid has been shown to inhibit the development of mammary tumors in mice, VIP may be a promoter of carcinogenesis (36).

Deletion of the N-terminus of VIP results in VIP receptor antagonists such as VIP^10–28 or neurotensin^6–11VIP^7–28 (VIPhybrid) (8). Subsequently (N-stearyl, Nle¹⁷)VIPhyb was developed, which was shown to bind with 100-fold high affinity than VIPhyb and to be neuroprotective (37). (SN)VIPhyb has been shown to inhibit the growth of 51 out of 56 cancer cell lines, including those derived from breast cancer, colon cancer, glioblastoma, leukemia, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and renal cancer (38). (SN)VIPhyb potentiated the cytotoxicity of taxol in breast cancer and of cisplatin, doxorubicin, gemcitabine, irinotecan or vinorelbine in colon cancer (39,40); (SN)VIPhyb inhibited the growth of lung cancer cells, whereas VIP stimulated proliferation. Because VIP was detected in extracts of lung cancer cells (38), it may function as an autocrine growth factor in lung cancer.

GRP/NMB

Gastrin releasing peptide is derived from the 148 amino acid preproGRP (10). High levels of GRP (27 amino acids) are present in SCLC (41, 42). Also, SCLC has elevated levels of creatine kinase and neuron specific enolase (43). Antibodies to GRP inhibit the growth of SCLC in vitro and in animal models in vivo (44). Elevated levels of proGRP^31–98 have been detected in the serum of SCLC patients (45). NMB (10 amino acids) is derived from the 121 amino acid preproNMB (10). Moderate levels of NMB are present in SCLC cells (46). GRP and NMB function as autocrine growth factors in lung cancer.

When GRP is secreted from SCLC cells it binds with high affinity to BB₂R (Table II). BB₂R is a type I (Rhodopsin) GPCR that has 384 amino acids and interacts with G_q, leading to PI turnover. The BB₁R has 390 amino acids, whereas the orphan BB receptor subtype 3 (BRS-3) had 399 amino acids; BB₁R and BRS-3 have approximately 50% sequence homology with BB₂R. BB₂R binds BB and GRP with high affinity whereas BB₁R binds NMB with high affinity (Table II). GRP has 9 of the 10 C-terminal amino acids as does BB, whereas NMB has 7 of the same 10 amino acids as BB. BRS-3 does not bind BB, GRP, or NMB with high affinity; however, BA1 binds with high affinity to BB₁R, BB₂R, and BRS-3 (10). BW2258U89 is a selective peptide antagonist for BB₂R, whereas PD168368 is a nonpeptide antagonist for BB₁R (47). ML-18 is a nonpeptide antagonist which prefers BRS-3 over BB₁R or BB₂R (48).

Table II.

Binding of BB-like peptides

Addition Ligand	IC50, nM
	BB2R	BB1R	BRS-3
BB	1.5 ± 0.2	150 ± 18	>10,000
GRP	2 ± 0.3	190 ± 22	> 10,000
NMB	170 ± 15	1.0 ± 0.3	> 10,000
PD168368	9000 ± 1150	1.8 ± 0.3	> 10,000
ML18	10,000 ± 1450	>10,000	3,000 ± 250
BW2258U89	10 ± 3	>10,000	>10,000

The IC₅₀ (nM) to inhibit specific ¹²⁵I-BA1 binding to NCI-H1299 cells stably transfected with BB₂R, BB₁R, or BRS-3 is indicated. The mean value ± S.D. of 3 experiments each repeated in quadruplicate is indicated. The structures of BB, GRP, and NMB, with sequence homologies relative to BB underlined, are:

BB: Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂

GRP: Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH₂.

NMB: Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH₂.

BBR agonists, but not antagonists, are internalized after binding to lung cancer cells at 37 ^oC. Initially, the GRP–BB₂R complex is internalized into endosomes, then perinuclear vesicles and subsequently the ligand is localized to lysosomes whereas the BB₂R is recycled to the plasma membrane (49). Because the C-terminal is essential for high affinity binding of BB to the BB₂R, drugs can be coupled to the N-terminal of BB which are cytotoxic for cancer cells. Camptothecin-linker2 (CPT-L2)–BA1 conjugates bind with high affinity to BB₁, BB₂R, and BRS-3. Surprisingly, CPT-L2–BA1 conjugates bind with approximately an order of magnitude greater affinity than BA1 (Table III). This suggests that CPT may interact with additional hydrophobic sites near the BA1 binding site. Using NCI-H1299 lung cancer cells transfected with BB₂R, BB₁R, or BRS-3 approximately 43, 34, and 42% of the added ¹²⁵I-CPT-L2–BA1 was internalized. After internalization into the lysosome, CPT is released which inhibits topoisomerase 1 (50). CPT-L2–BA1 inhibited the proliferation of breast cancer, colon cancer, gastric cancer, glioblastoma, leukemia, lung cancer, neuroblastoma, pancreatic cancer and prostate cancer cell lines. BB conjugated to paclitaxel, doxorubicin, magainin, or marine toxins and siRNA-specific for BB₂R are cytotoxic for cancer cells (51–56).

Table III.

Binding of BA1-like peptides

Addition Ligand	IC50, nM
	BB2R	BB1R	BRS-3
BA-1	3.2 ± 0.12	7.4 ± 0.5	2.5 ± 0.1
CPT-L2-BA1	0.12 + 0.01	0.35 +0.03	0.31 + 0.08

The IC₅₀ (nM) to inhibit specific ¹²⁵I-BA1 binding to NCI-H1299 cells stably transfected with BB₂R, BB₁R, or BRS-3 is indicated. The mean value ± S.D. of 3 experiments each repeated in quadruplicate is indicated. The structures of BA1 and CPT-L2-BA1, with sequence homologies relative to BA1 underlined, are:

BA1: D-Tyr-Gln-Trp-Ala-Val-β-Ala-His-Phe-Nle-NH₂

CPT-L2-BA: CPT-L2-D-Ser- D-Tyr-Gln-Trp-Ala-Val-β-Ala-His-Phe-Nle-NH₂

BB₁R, BB₂R, and BRS-3 regulate transactivation of EGFR in cancer cells (57–59); the GPCR interacts with G_q causing PI turnover. The IP₃ released causes elevation of cytosolic Ca²⁺ within seconds whereas the DAG released activated PKC. BA1 addition to NSCLC cells increases EGFR tyrosine phosphorylation within 1 min and EGFR transactivation is blocked by PP2, NAC, or GM6001 (Fig. 1). Because TGF-α is released from the NSCLC cells, EGFR transactivation is blocked by gefitinib or DPI. Also, the transactivation regulated by BB₁R, BB₂R, and BRS-3 is blocked by the GPCR antagonists PD168368, BW2258U89, and ML-18, respectively.

Addition of GRP or NMB to NSCLC cells increases their proliferation, which is antagonized by BW2258U89 or PD168368, respectively (13,14). Also, BW2259U89, PD168369, or ML-18 inhibit basal proliferation of NSCLC cells in vitro. Using mouse models in vivo, BW2258U89 or PD168368 inhibit xenograft growth in a cytostatic manner in that is drug administration is discontinued the tumors rapidly regrow. In contrast, gefitinib is a cytotoxic agent that causes cancer cell apoptosis in vitro and in vivo; this cytotoxicity is potentiated by PD168368 or ML-18 (48,57). The results indicate that GPCR antagonists are synergistic with gefitinib at inhibiting NSCLC proliferation.

BBRs are present in numerous cancers. BB₂R are most abundant in breast, colorectal, glioblastoma, head & neck, lung, ovarian, prostate and renal cancers (58–60). BB₁R is detected in intestinal carcinoids whereas BRS-3 is present in lung and renal cancers. BB₁R, BB₂R, and BRS-3 mRNAs were detected in 11/13 lung cancer cell lines (61). Prostate cancer tumors in patients were detected using either a ⁶⁴Cu-BB^6–14 (62) or ^99mTc-BB^2–14 analog (63). Breast cancer tumors in patients were detected using a ^99mTc-RGD-BB analog (64). It remains to be determined if radiolabeled BB analogs will be useful for the early detection of cancer.

SUMMARY

GPCRs of VIP and BB are important for the growth of cancer cells. VPAC1, VPAC2, and PAC1 activate adenylyl cyclase, leading to elevation of cAMP. cAMP-stimulated PKA leads to phosphorylation of CREB and altered gene expression. VIP and PACAP stimulate the growth of lung cancer cells; growth of NSCLC can be inhibited by VIP-cytotoxic analogs or VIPR antagonists such as VIPhyb. Radiolabeled VIP and PACAP analogs may be useful for imaging patients’ tumors. Also, PAC1 regulates PI turnover and transactivation of EGFR.

BB₁R, BB₂R, PAC1, and BRS-3 cause PI turnover and subsequent elevation of cytosolic Ca²⁺ and activation of PKC; this leads to phosphorylation of ERK and altered gene expression. NMB, GRP, and BA1 stimulate the growth of lung cancer cells, which can be inhibited by GPCR antagonists such as PD168368, BW2258U89, or ML-18. CPT-BA1 analogs are cytotoxic for cells which have BBRs. Also, BB₁R, BB₂R, and BRS-3 regulate the transactivation of EGFR. EGFR increases cancer proliferation through the Ras–Raf–MEK–ERK pathway and increases cellular survival through the PI3K–Akt–Tor pathway. The transactivation of EGFR is impaired by BBR antagonists and the TKI gefitinib. Gefitnib is used to treat patients with NSCLC who have EGFR mutations and have failed chemotherapy (65, 66). Our results indicate that BB₁R, BB₂R, and BRS-3 antagonists potentiate the cytotoxicity of gefitinib.

New ligands that interact with BBRs have been developed. Previously, BB receptor agonists were utilized to image tumors; however, better results are being obtained using antagonists that have fewer adverse effects and better pharmacokinetics (67). For example, ⁶⁸Ga-sarabesin 3 binds with high affinity to prostate cancer cells and imaged prostate tumors in mice (68). Also, Bantag-1, a new BRS-3 antagonist, has been shown to inhibit lung cancer proliferation (69). Traditionally SCLC is treated with chemotherapy as well as radiation therapy, and NSCLC is treated with chemotherapy; however, the 5-year survival rate is 16%. With over 150,000 U.S. citizens dying annually from lung cancer, new therapeutic approaches are needed. Peptide GPCRs are important targets for development of new drugs for treatment of cancer patients.