No NK-2R or NK-3R antagonist has been approved for clinical practice
[12], although several NK-2R or NK-3R antagonists have been tested in clinical trials
[14]. Unfortunately, although NK-2R/NK-3R antagonists were safe, these trials were abandoned due to the lack of efficacy. The ineffective results could also have been due to a non-appropriate selection of patients and endpoints in clinical trials and a lack of knowledge regarding the molecular interaction between tachykinin ligands and NK receptors
[15]. Moreover, the ineffective effects could also be due to a non-appropriate selection of patients and endpoints in clinical trials and to a lack of knowledge regarding the molecular interaction between tachykinin ligands and NK receptors
[15]. NK-2R antagonists (e.g., nepadutant (MEN-11,420) blocked tumor cell proliferation promoted by NKA in breast carcinoma
[99]. Metastatic and non-metastatic breast cancer cell proliferation was blocked by inhibiting the action of SP, at NK-2R, with NK-2R antagonists (GR-159,897); however, this action was less prominent than that observed with NK-1R antagonists (RP-67,580)
[100]. This finding suggests that breast cancer cells respond differently to NK-2R and NK-1R antagonists. Moreover, these cells reacted differently to NK-2R and NK-1R agonists (e.g., SP)
[100].
4.2. Calcitonin/Calcitonin Gene-Related Peptide Family
4.2.1. Adrenomedullin
AM ligand/receptor overexpression occurs in colonic cancers, and antibodies directed against both targets decreased tumor growth
[162][163][164][244][245][246][247]. The peptide fragment AM
22–52 [212], polyclonal antibodies against AM
[175][248] or its receptor
[247], monoclonal antibodies
[249], and small interfering RNAs
[215] regulate AM expression and actions
[166]. Small molecules (e.g., 16311, a negative AM modulator; 145425, a positive AM modulator) also regulate the effects mediated by AM. For example, 145425 reduced tumor burden, colon weight/length ratio, and tumor growth in mice
[166]. AM promotes cell proliferation and survival, alters the cell phenotype more aggressively, and increases vascularization
[250]. Tumors are, in general, the main source of excessive AM production since AM levels returned to normal levels when the tumor was removed after surgery
[251]. The blockade of AM receptors or lowering AM amounts are antitumor strategies to decrease the tumor mass
[133]. Many preclinical studies have shown reduced tumor cell proliferation, metastasis, and angiogenesis after applying AM receptor antagonists, AM receptor interference, or AM-neutralizing antibodies
[250]. Thus, a cocktail of antibodies directed against CLR and RAMP2/3 exerted an antitumor effect against glioblastoma, mesothelioma, and lung and colon cancer, and peptide antagonists such as AM
22–52 also showed an antitumor action against melanoma, mesothelioma, and renal, ovarian, breast and pancreatic cancer
[175][247][252].
Tumor development is highly dependent on the formation of both lymphatic (lymphangiogenesis) and blood (angiogenesis) vessels from pre-existing ones; these mechanisms are regulated by AM and VEGF, which are released from cancer and tumor stroma cells (e.g., endothelial cells, pericytes, fibroblasts, and macrophages)
[250]. Thus, the inhibition of both angiogenesis and lymphangiogenesis mechanisms is a sound antitumor strategy. The AM/RAMP2 system controls vascular integrity. It has been reported that the deletion of RAMP2 favored vascular permeability and the formation of pre-metastatic niches in distant organs by altering the vascular structure and promoting inflammation
[253]. As a result, the AM/RAMP2 system regulates vascular integrity, and this system could be a promising antitumor therapeutic target to block metastasis. AM plays an essential role as a crosstalk agent integrating mast and tumor cell communication
[254]. AM favors the release of beta-hexosaminidase or histamine from human mast cells, which are also involved in angiogenesis; this was inhibited with anti-AM monoclonal antibodies
[254].
The AM/RAMP2 system is involved in tumor angiogenesis; liver metastasis (PAN02 pancreatic cancer cells were administered into the spleen) increased in vascular endothelial cell-specific RAMP2 knockout mice, and liver metastasis was suppressed in RAMP3
−/− animals in which the number of podoplanin-positive cancer-associated fibroblasts decreased in the periphery of tumors at metastatic sites
[216]. RAMP3 deficiency cancer-associated fibroblasts inhibited cell proliferation/migration and metastasis, and the activation of RAMP2 in RAMP3
−/− mice blocked tumor growth and metastasis
[216]. Moreover, podoplanin upregulation in RAMP2
−/− animals augmented malignancy, and podoplanin downregulation in RAMP3
−/− mice decreased malignancy
[216]. The observation means that RAMP2 activation and RAMP3 inhibition can suppress metastasis, and that deficiency of the AM/RAMP3 system inhibited metastasis via the modification of cancer-associated fibroblasts. Acylated truncated AM/AM2 analogs of 27–31 residues showed a potent antagonistic action toward CLR/RAMP1, and non-acylated analogs showed minimal activity
[255].
4.2.2. Adrenomedullin 2
AM2 is involved in vascular remodeling processes and angiogenesis; hence, the peptide is an important target for developing angiogenesis-based antitumor strategies
[256]. AM2 increases tumor blood perfusion and promotes quiescent endothelial cells to proliferate by restraining endothelial cell response to VEGF; hence, the excessive vessel sprouting is blocked, and the vascular lumen is increased
[257]. AM2 also favors the formation of a signaling complex containing CLR/β-arrestin1/Src in endothelial cells and promotes its internalization into the cytoplasm via a clathrin-dependent manner to activate downstream ERK1/2 pathway; this action was not inhibited by endothelial cell contact
[257].
AM2 blockade with neutralizing antibodies/antagonist peptides inhibited the growth of tumor cells by promoting apoptosis, Bcl2/Gli1 downregulation, and caspase-8 cleavage
[189]. Significantly, administering anti-AM2 monoclonal antibodies not only inhibited tumor growth but also increased the antitumor activity of temozolomide
[180]. By using anti-AM2 antibodies, ERK1/2 phosphorylation was inhibited in endothelial cells and, in the same cells, a decreased expression of vascular endothelial cadherin and dissociation of vascular endothelial cadherin/vascular endothelial growth factor receptor 2/phosphoinositide 3-kinase complex occurred, leading to the internalization and phosphorylation of vascular endothelial growth factor receptor 2 and the blockade of the PI3K/Akt pathway
[256].
4.2.3. Amylin
The p53 family promotes tumor suppression, and deletion of the ΔN isoforms of p63 or p73 led to metabolic reprogramming and regression of p53-deficient tumors via upregulation of the
AMY gene
[237]. AMY is involved in tumor regression and, via the CT receptor/receptor activity modifying protein 3, promotes apoptosis, induces reactive oxygen species, and blocks glycolysis
[237]. Pramlintide, a synthetic analog of AMY, stimulated tumor regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers
[237]. Pramlintide also exerted an antiproliferative action against colorectal cancer cells, and its coadministration with classic chemotherapeutics increased cytotoxicity
[258].
4.2.4. Calcitonin Gene-Related Peptide
CGRP signaling, through the CT receptor, increased chemotherapy resistance and stem cell properties in acute myeloid leukemia, and olcegepant, a CGRP antagonist, decreased key stem cell properties and leukemic burden
[140]. The CGRP/CT receptor system could be an antitumor target against acute myeloid leukemia. Moreover, the CGRP8-37 peptide antagonist exerted an antitumor action against prostate cancer
[259].
CGRP augmented cytotoxic CD8
+ T cell exhaustion, limiting their capacity to eliminate melanoma cells
[260]. CGRP antagonism of the receptor RAMP1 or pharmacological silencing of nociceptors decreased tumor-infiltrating leukocyte exhaustion and B16F10 melanoma cell growth
[260]. Thus, reducing CGRP release from tumor-innervating nociceptors is a valuable strategy to improve antitumor immunity via eliminating the immunomodulatory actions exerted by CGRP on these cytotoxic T cells
[260].