5. EORs involved in diseases
Some EORs may show no or very low expression in healthy tissues. Nevertheless, they can show high expression in cancer and diseased tissues, which might represent such kinds of EORs as potential biomarkers for pathogenesis.
5.1 Breast Cancer
OR2B6, a tissue-specific EOR in breast cancer, has been found 73% and 80% expression of OR2B6 in breast carcinoma cell lines and carcinoma tissues respectively. The upregulated expression of OR2B6 was found in blood platelets of tumors from breast cancer patients. OR2B6 can also express mutually with a histone gene HIST1H2BO and build a fusion transcript together in carcinoma tissues. However, healthy tissues hardly show any expression of OR2B6 that makes it to be a probable biomarker for breast cancer. OR2B6 can also express in several carcinoma tissues, but not as remarkable as breast carcinoma tissues (Weber et al., 2018a). OR2W3 and OR2T8 are also highly up-regulated. These three EOR genes are “over-expressed” in breast cancer tissues (Masjedi, Zwiebel & Giorgio, 2019).
5.2 Bladder Cancer
Bladder cancer tissues show a significant expression of OR10H1 compared to the normal bladder. The triggering of this receptor can change the morphology of cytoskeletons that can be identified β-Catenin, T-cadherin, and β-actin staining. Stimulation by sandranol blocks cell migration and proliferation, implies cell cycle arrest and leads to a limited extent- apoptosis. Sandranol inhibits adenylyl cyclase and thus reduces cAMP levels which evoke an increase of intracellular Ca2+ concentration (Weber et al., 2018b).
5.3 Lung cancer
RSK1 silencing increases tumor metastasis in non-small-cell lung cancer (NSCLC) tissues in humans. This cell shows very strong positivity about the expression of OR2J3. This receptor also can start the release of Ca2+ from intracellular Ca2+ stores (Kalbe et al., 2017). Functional imaging and immunohistochemical studies show quite stable and high expression of OR51E1 in lung cancer cells. Due to the extensive membrane localization, OR51E1 can be considered as a novel therapeutic target against available Somatostatin receptors (SSTRs). Moreover, some tumor patients do not respond to SSTRs based diagnosis (Giandomenico et al., 2013)
5.4 Colorectal Cancer
In 2017, Weber et al. found extraordinarily over-expression of OR51B4 in colorectal cancer tissues confirmed by shRNA mediated knockdown. In HCT116 cells, Troenan can stimulate anti-proliferation, anti-migration, and pro-apoptosis by PLC activation and intracellular calcium level changes. This results in phosphorylation levels changes of p38, mTOR and Akt kinases (Weber et al., 2017). Cancer initiating cells (CICs) in colon expresses OR7C1, which results in higher tumorigenicity. Peptide specific cytotoxic T lymphocyte (CTL) antigen for OR7C1 is toxic to CICs (Morita et al., 2016).
5.5 Myelogenous leukemia
By next-generation sequencing, a recent study shows that chronic myelogenous leukemia (CML) cell express OR at a high rate, specifically OR51B5. Isononyl alcohol activates OR51B5 and increase intracellular Ca2+ level in acute myelogenous leukemia (AML) patients. OR51B5 can inhibit cell proliferation in both AML and CML patients by reducing the phosphorylation of p38MAPK (Manteniotis et al., 2016b).
OR2AT4 can increase the phosphorylation of p38-MAPK that leads to leukemia. This receptor expresses highly in human myelogenous leukemia (Manteniotis et al., 2016a)
5.6 Hepatic diseases
OR1A2, which is paralogous to OR1A1, is a potentially expressed olfactory receptor in hepatic cancer cells. (-)citronellal activates OR1A2 which increases cytosolic Ca2+ level via the cAMP-dependent pathway and reduces cell proliferation by p38MAP phosphorylation (Meβberg et al., 2015). As a null variant, OR1B1 gene influences liver cell metabolism by reducing serum cholinesterase activity. It aids to effect significantly in liver autoimmune disease (Koyano et al., 2008).
5.7 Retinitis Pigmentosa
Definite mutation in OR2W3 gene is highly related to an ocular disease called Retinitis pigmentosa (RP) (Ma et al., 2015). RP is an inherited autosomal dominant retinal disease, which is rare, reported only one of 3000 to 5000 peoples (Zhang & Huang, 2015). OR2W3 is located in the photosensitive outer membrane of cone cells. As it does not fuse with Trim58 transcript, it can be concluded that it possibly has a physiological function in the human retina (Sharon, Kimchi & Rivolta, 2016).
5.8 Neurological Disorders
Neurodegenerative and neuropsychiatric disorders evaluation in recent days proves the relation with dysregulated OR gene expression. Several ORs (OR2L13, OR1E1, OR2J3, OR52L1, and OR11H1) have been identified to be down-regulated in the early stages of Parkinson’s disease pathogenesis (Garcia-Esparcia et al., 2013; Grison et al.,2014), which may lead to their undeniable importance in the development of the disease. Patients samples with Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and progressive supra-nuclear palsy present differentially regulated OR gene expression (Ansoleaga et al.,2013). Among the verified ORs in AD patients, half showed altered expression in the cortical region. OR11H1 supposed to be up-regulated, while OR4F4, OR52L1, and OR10G8 expression are reduced and linked up with disease development (Ansoleaga et al., 2013; Woodward et al., 2017).
Downregulated ORs in cerebral regions leads to chronic schizophrenia (Ansoleaga et al., 2015) and traumatic brain injury (Zhao et al., 2013). For biomarker analysis, the detection of both OR4M1 and OR11H1 can have a potential diagnostic feature in the near future (Maßberg & Hatt, 2018). Down-regulation of OR2L13, OR2T33, OR2J3, OR52L1, OR10G8, OR11H1 and OR4F4 in frontal cortex has been detected in the early stages in Parkinson’s disease (Grison et al., 2014). Olfactory receptor gene cluster on 14q11.2 region containing OR4M1, OR4N2, OR4K2, OR4K5, and OR4K1 shows modifications in their expression at the earlier age of Alzheimer’s disease (Ansoleaga et al.,2013).
5.9 Urological disorders
OR51E1 and OR51E2 are the most distributed and expressed ectopic OR. They make themselves noble biomarkers by expressing higher in cancer tissues than healthy tissues. They were first introduced as a significant OR, expression restricted to prostate adenocarcinoma and named as PSGR (OR51E2) and PSGR2 (OR51E1) (Xia et al., 2001; Xu et al., 2006). PSGR locates in chromosome 11p15 encoding 320 amino acid-containing proteins (Xu et al., 2000). Its mRNA level, correlated with prostate-specific antigen (PSA), raises remarkably prostate intraepithelial neoplasia (PIN) and Prostate cancer (PCa) (Xu et al., 2006). As it is available in human urine sediment, it can be used as an alternative biopsy for prostate cancer (Rigau et al., 2010). According to Cao et al., PSGR can boost up cell proliferation invasion and later on metastasis in prostate cancer (Cao et al., 2015). The elevated expression refers to an early alteration of PCa while low expression reveals poor prognosis. Both OR51E1 and OR51E2 can be linked with PCa marker alpha-methyl-CoA racemase (AMACR) (Wang et al., 2006). Therefore, a dual marker can be a good identifier to ensure the development of prostate cancer. Besides, PSGR can be fused up with erythroblast transformation specific (ETS) transcription factor (ETV-1) chimerically and show important positive effects on the elevation of prostate cancer (Weng et al., 2005; Barros-Silva et al., 2013).
5.10 Others
OR51E1 can also be a potential biomarker for the detection of somatostatin receptor-negative lung carcinoids (Giandomenico et al., 2013) and small intestinal neuroendocrine carcinomas (Leja et al., 2009; Cui et al., 2013). A recent study mentioned that peptide derived from OR51E2 can behave as tumor-associated antigen (TSA), detected by CD8+ T-cells, in various cancer cells including melanoma (Gelis et al., 2017).

6. Possible mechanisms of EORs

Even EORs are not involved in smell and neural perception; they share the same structure with ORs in the nasal epithelium as well as their mechanisms. The signaling of EORs is hypothesized by the fragmentary involvement of cAMP, recommending heterotrimeric G-protein (Golf protein) as a strong stimulatory that widely expressed in human tissues (Flegel et al., 2013; Busse et al., 2014).
6.1 The initial process for EORs activation
When a ligand binds to the OR, the 7 transmembrane receptor initiates and converts its conformation based on the interaction between GPCR and G-protein. The Gα subunit presumes a triggered conformation upon GTP-binding and dissociates from receptor and Gβγ. When GPCR activates, Gα releases from GDP. “Empty pocket” of G-protein and the receptor bridges with a high-affinity complex, which can be described with “action at a distance” hypothesis elaborated by Oldham & Hamm (2008). The conversion of GTP from GDP results in dissociation of Gβγ dimer from Gα that further starts intracellular signaling as “second messenger” (Sprang, 2007). This second messenger can initiate or inhibit other elements of cell mechanisms. For instance, Phospholipase C enzyme can hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG can actuate protein kinase C isoforms. IP3 can connect with receptors which lead to calcium release in the cytosol. A large number of the second messenger including cyclic AMP, cyclic GMP, calmodulin, and kinases can be modulated by G proteins. When GTP hydrolyzes into GDP by GTPase activity of the Gα subunit, inactive Gαβγ is formed by the re-association of Gα-GDP and Gβγ. Evidence implicates that there is no physical disassociation of G-protein from the complex (Frank et al., 2005; Digby et al., 2006).
An activated G-protein can co-localize with regulatory factor Resistance to inhibitors of cholinesterase 8B (Ric8B) in olfactory sensory neurons. Although there is no solid information for specific functional appearance, there are some limited proof and proposed data of G-protein subunit activation in cancer cells (Sanz et al., 2014). ORs require definite cofactors to develop membrane localization of receptors. The important co-factors are Receptor transporter proteins 1 and 2 (RTP1 and RTP2) and receptor expression enhancing protein (REEP1). They are found in neurons cytoplasm doing down-regulation of brain’s signaling molecules (Krautwurst, Yau, & Reed, 1998; Abaffy, Matsunami, & Luetje, 2006; Li & Matsunami, 2011; Peterlin, Firestein & Rogers, 2014)
6.2 cAMP-induced calcium flux
Most of the ectopic ORs trigger a cAMP-induced calcium influx. It generally happens from outside the cells representing the canonical pathway cascade in olfactory sensory neurons (OSNs). The necessary subunits expressions to build the canonical heterotetrameric cyclic nucleotide-gated (CNG) channel (CNGA2, CNGA4, and CNGB1), particularly CNGA2, has not been identified in most tissues of the human body. CNG channel is a very important channel in peripheral human tissues and cells (Flegel et al., 2013). CNGA1, mainly activated by cGMP along with cAMP, is the native rod protein capable of forming functional homomeric channels. CNGA1 can perform as a CNG channel (Kaupp et al., 1989). CNGA3 channel is a cone photoreceptor native to sperm cells. Along with the CatSper channel, CNGA3 expressed functionally indicating its possible involvement in OR-mediated sperm chemotaxis (Busse et al., 2014; Maßberg et al., 2015)
Though the Ca2+ entering channels are still mostly unidentified, some researches have shown the involvement of TRP channels, CRAC channels, voltage-gated L-type Ca2+channels, or spermatozoa-specific CatSper channels (Brown et al.,2019). There is a possibility of determining Ca2+influx through TRPM family members by using particular channel blockers 2-APB (Kalbe et al., 2016b; Flegel et al., 2016; Manteniotis et al., 2016b).
Intracellular Ca2+ increment in human airway smooth cell is induced by OR1D2 and OR2AG1 through a cAMP-dependent pathway. OR1A1 induces cAMP response element-binding protein (CREB) without cAMP induction and intracellular Ca2+.
6.3 MAPK downstream cascade
Ectopic OR activation can lead to regulate downstream protein kinase cascades, precisely MAPK, in various cellular signaling dependent or independent of the canonical pathway. These protein kinases presume to be the main downstream modulators of several cellular processes (Kim et al., 2015; Gelis et al., 2016)
These ORs mediated signaling in physiological systems are distinctly relied on the OR ligands structure and concentration, morphology and biochemistry of the regarding cellular systems, the heterotrimeric G protein subunits, and the involvement of other less regulatory scaffold proteins(Rodriguez et al., 2014; Wiese et al., 2015; Wu et al., 2015). By reducing early phosphorylation of p38 mitogen-activated protein kinases (p38-MAPK), OR2AT4 can hinder cell growth and through the phosphorylation of p44/42-MAPK can decrease cell apoptosis in acute myelogenous leukemia (AML) patient (Manteniotis et al., 2016a).
6.4 Others
Some ORs can activate the tyrosine kinase Src (sarcoma) signaling pathway. Without activating any G-protein, this cascade can raise the Ca2+ level in the cell via transient receptor potential channel V6 (TRPV6). Some evidences show that some specific ligands such as β-ionone can trigger PI3K/AKT downstream signaling via Gβγ stimulation. OR51E1 shows involvement in AR-mediated signaling through Src kinase (Spehr et al., 2011; Maßberg et al.,2016).
In essence, ORs activated by their specific odorants can transduce intracellular signal cascade by several mechanisms.