Terry W. Moody

The bombesin (Bn) receptor family [Gastrin-releasing peptide (GRPR/BB2R) and Neuromedin B receptors (NMBR/BB1R)] are G-protein coupled receptors (GPCR’s) with potent growth effects on normal tissues/numerous cancers, often by transactivating the ErbB receptor-tyrosine kinase (RTK) family. Whereas GRPR stimulation transactivates ErbB RTKs EGFR, HER2, and HER3 in non-small cell lung-cancer (NSCLC) cells, its effects on HER4 are unknown. This study was designed to address this question. Of 12 NSCLC’s studied, 75% had HER4 mRNA expression and Western-Blotting. NCI-H522 and NCI-H661-cells had high levels of GRPR, HER4, and the HER4-ligand neuregulin (NRG1). Adding GRP to NCI-H522/NCI-H661-cells activated HER4, shown by its increased phosphorylation (P-HER4). The GRPR antagonists PD176252/BW2258U89 inhibited this increase. In NCI-H661-cells, GRP stimulated the formation of HER4-homodimers and HER2-HER4-heterodimers. Adding GRP to these NSCLC-cells increased P-ERK/P-AKT, which was inhibited by siRNA-HER4, PD176252, and ibrutinib, as well as N-acetylcysteine and Tiron, which reduce reactive-oxygen species (ROS). GRP increased secretion of NRG1 from NSCLC-cells, and NRG1 increased P-HER4 and P-ERK, which were impaired by ibrutinib. GRP and NRG1 stimulated proliferation of NSCLC-cells, which was inhibited by PD176252, siRNA-HER4, or ibrutinib and which was mediated by MAPK, not AKT/PI3K, activation. These results show GRPR activation results in HER4 transactivation in a ROS-dependent manner, which stimulates NSCLC-growth through a MAPK-mediated mechanism.

Bombesin receptor subtype-3 (BRS-3) is a type 1 G-protein-coupled receptor (GPCR). BRS-3 is an orphan GPCR that is structurally related to neuromedin B and gastrin-releasing peptide receptors. When activated, BRS-3 causes phosphatidylinositol turnover in lung cancer cells. BRS-3 stimulates tyrosine the phosphorylation of the epidermal growth-factor receptor (ErbB1); however, it is unknown whether it transactivates ErbB2/HER2. Adding the nonpeptide BRS-3 allosteric agonist MK-5046 or the peptide agonist BA1 to the lung cancer cell line NCI-H727 or to BRS-3-transfected NCI-H1299 lung cancer cells increased the tyrosine phosphorylation of HER2/ERK2. This increase was antagonized by the BRS-3 peptide antagonist Bantag-1 and the small-molecule BRS-3 antagonist ML-18. The increase in HER2/ERK phosphorylation caused by MK-5046 was inhibited by the ROS inhibitors N-acetylcysteine and Tiron (superoxide scavengers). Adding MK-5046 to lung cancer cells increased reactive oxygen species, which was inhibited by NAC or Tiron. MK-5046 and BA1 increased non-small lung cancer cell (NSCLC) colony formation, whereas Bantag-1/ML-18 inhibited proliferation. These results indicate that in lung cancer cells, the activation of BRS-3 regulates HER2 transactivation in an ROS-dependent manner, which can mediate tumor growth. These results raise the possibility that the use of HER2-inhibiting compounds alone or in combination with other agents could represent a novel approach to the treatment of these tumors.

The ErbB RTKs (EGFR, HER2, HER3, and HER4) have been well-studied in cancer. EGFR, HER2, and HER3 stimulate cancer proliferation, principally by activating the phosphatidylinositol-3-kinase and extracellular signal-regulated kinase (ERK) pathways, resulting in increased cancer cell survival and proliferation. Cancer cells have high densities of the EGFR, HER2, and HER3 causing phosphorylation of tyrosine amino acids on protein substrates and tyrosine amino acids near the C-terminal of the RTKs. After transforming growth factor (TGF) α binds to the EGFR, homodimers or EGFR heterodimers form. HER2 forms heterodimers with the EGFR, HER3, and HER4. The EGFR, HER2, and HER3 are overexpressed in lung cancer patient tumors, and monoclonal antibodies (mAbs), such as Herceptin against HER2, are used to treat breast cancer patients. Patients with EGFR mutations are treated with tyrosine kinase inhibitors, such as gefitinib or osimertinib. Peptide GPCRs, such as NTSR1, are present in many cancers, and neurotensin (NTS) stimulates the growth of cancer cells. Lung cancer proliferation is impaired by SR48692, an NTSR1 antagonist. SR48692 is synergistic with gefitinib at inhibiting lung cancer growth. Adding NTS to lung cancer cells increases the shedding of TGFα, which activates the EGFR, or neuregulin-1, which activates HER3. The transactivation process is impaired by SRC, matrix metalloprotease, and reactive oxygen species inhibitors. While the transactivation process is complicated, it is fast and occurs within minutes after adding NTS to cancer cells. This review emphasizes the use of tyrosine kinase inhibitors and SR48692 to impair transactivation and cancer growth.

Peptide G protein–coupled receptors (GPCRs) for pituitary adenylate cyclase activating polypeptide (PACAP) regulate the growth of non-small cell lung cancer (NSCLC) cells. PACAP binds with high affinity to PAC1, which causes transactivation of receptor tyrosine kinases (RTK) for the EGFR and HER2 but its effect on HER3 is unknown. Using 3 NSCLC cell lines (NCI-H358, NCI-H441, and Calu-3), proteins for EGFR, HER2, HER3, and PAC1 were detected. The increase in EGFR tyrosine phosphorylation caused by PACAP was blocked by the EGFR tyrosine kinase inhibitor (TKI) gefitinib, or PACAP(6-38), a PAC1 antagonist. The increase in HER2 tyrosine phosphorylation caused by PACAP was inhibited by trastuzumab, a monoclonal antibody (mAb) for HER2, or PACAP(6-38). The increase in HER3 tyrosine phosphorylation caused by PACAP was inhibited by HER3 mAb3481 or PACAP(6-38). Immunoprecipitation experiments indicated the PACAP addition to Calu-3 cells resulted in the formation of EGFR/HER3 and HER2/HER3 heterodimers. Addition of the HER3 agonist neuregulin (NRG)-1 increased HER3 tyrosine phosphorylation in non-small-cell lung cancer (NSCLC) cells. PACAP or NRG-1 increased the proliferation of NSCLC cells, whereas PACAP(6-38), gefitinib, trastuzumab, or mAb3481 inhibited proliferation. The results indicate that PAC1 regulates the proliferation of NSCLC cells as a result of transactivation of the EGFR, HER2, and HER3.

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 (BBRs), 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 cyclic AMP response element binding protein (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.

Neuropeptide G protein-coupled receptors (GPCRs) are overexpressed on numerous cancer cells. In a number of tumors, such as small cell lung cancer (SCLC), bombesin (BB) like peptides and neurotensin (NTS) function as autocrine growth factors whereby they are secreted from tumor cells, bind to cell surface receptors and stimulate growth. BB-drug conjugates and BB receptor antagonists inhibit the growth of a number of cancers. Vasoactive intestinal peptide (VIP) increases the secretion rate of BB-like peptide and NTS from SCLC leading to increased proliferation. In contrast, somatostatin (SST) inhibits the secretion of autocrine growth factors from neuroendocrine tumors (NETs) and decreases proliferation. SST analogs such as radiolabeled octreotide can be used to localize tumors, is therapeutic for certain cancer patients and has been approved for four different indications in the diagnosis/treatment of NETs. The review will focus on how BB, NTS, VIP, and SST receptors can facilitate the early detection and treatment of cancer.

While peptide antagonists for the gastrin releasing peptide receptor (BB2R), neuromedin B receptor (BB1R) and bombesin receptor subtype-3 (BRS-3) exist, there is a need to develop nonpeptide small molecule inhibitors for all three BBR. The bombesin agonist (BA)1 binds with high affinity to the BB1R, BB2R and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB2R, BB1R and BRS-3 with similar affinity (Ki = 1.4-10.8 uM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells which have BBR. The results indicated that AM-37 and ST-36 function as small molecule BBR antagonists.