FIGURE 5-13
String™ prediction protein analysis diagram576
The following are predicted targets for Hsa-mir-4274 (Figure 5-13):
PC4 and SFRS1-interacting protein (PSIP1) has growth factor, transcription factor and cofactor activity496,578. Biologically it is involved in the immune system processes. At a cellular level it is involved in cell-cell signalling, signal transduction and regulates transcription from the RNA polymerase 2 promoter. The immune system in a normally functional state identifies aberrant or ectopic cells and causes apoptosis and elimination of these cells. Alterations at various levels in the immune system may aid in survival of ectopic cells that progress towards endometriosis.
Zinc finger protein 41 (ZNF41) is a protein with KRAB box transcription factor activity and is involved in spermatogenesis496,578. This protein is also seen to regulate transcription from RNA polymerase 2 promoter affecting the cellular cycle.
Regulator of G-protein signalling 20 (RGS20) binds proteins and has small GTPase regulator activity496,578. Biologically it contributes to cellular dorso-ventral axis specification and is involved in heterotrimeric G-protein signalling pathways (G1/Gs α and Gq/G0 α) regulators of G protein signalling.
Rap guanine nucleotide exchange factor 2 (RAPGEF2) is a member of the PDZ domain containing guanine nucleotide exchange factor family496,578. They stimulate the exchange of guanyl nucleotides through the use of a GTPase. It has functions of protein binding, small GTPase regulator activity and guanyl-nucleotide exchange factor activity involved in mitosis, the transmembrane receptor protein tyrosine kinase signalling pathway, G-protein coupled receptor protein signalling pathway and the MAPKKK cascade526. Receptor tyrosine kinases serve as cell surface receptors for molecules such as growth factors and cytokines582 and are associated with regulation of cellular processes. Alterations in the functioning of tyrosine kinases are associated with malignancy development583 and have been used as potential targets for cancer prevention. Subtle effects of RAPGEF2 on the transmembrane receptor
protein tyrosine kinase signalling pathway could potentially contribute to the development and formation of a disease such as endometriosis. If through functional studies, it appears to be an important component in endometriosis development, it could be used as a therapeutic target for this disease too. The G-protein coupled receptor protein signalling pathway is related to various important cellular functions. Pertinent to my study is its regulation of inflammation and the immune system as well as being linked to tumour growth and metastasis584. In recent years this is another family of receptors that are targeted for novel therapeutics585. Once again we can see a pathway which when disrupted, is responsible for ectopic tumorigenic growth and metastatic potential. Extrapolated to endometriosis, a disease where ectopic tissue implants grows and can even “metastasise” to organs outside of the pelvis, one can potentially see the importance of the roles of such pathways. Again functional studies are necessary with the potential of novel targeted therapeutics for endometriosis. The MAPKKK cascade is linked to the antiapoptotic RAS cascade and protein which in my study has been identified as one of the upregulated markers in the serum of women with endometriosis (See Table 6-4 Section 6.4.7). Mutations in the RAS/MAPK cascades have been linked to endometrial cancers586, it is therefore not difficult to postulate that milder aberrations in the pathways caused by Hsa-mir-4274 could be linked to development of endometriosis which is itself not classified as a malignancy but has properties of cell implantation, growth survival and dissemination.
Long-chain-fatty-acid-CoA ligase 6 (ACSL6) is a protein with ligase and transporter activity496,578. It too is involved in the immune system and fatty acid metabolism processes.
Methenyltetrahydrofolate cyclohydrolase (MTHFD2L) has oxidoreductase and hydrolase activity496,578. It is involved in metabolic processes which are purine and pyrimidine based and has a role in the amino acid biosynthetic processes within the cell.
Lysyl oxidase homolog 4 (LOXL4) has oxidoreductase, serine-type peptidase and receptor activity578. Biologically it is implicated in macrophage activation and the cellular defence response and has a role in proteolysis, cell-cell adhesion, signal transduction and extracellular transport496. It is observed in the processes involving the neurological system as a negative regulator of apoptosis. LOXL4 loss of expression has been seen in the development of bladder tumours, with its re-introduction decreasing the ability of the tumour to form colonies587. They can therefore function as tumour suppressor genes and inhibit the Ras/ERK (extracellular signal-regulated kinase) pathway in cancers. If LOXL4 acts in a similar way in endometriosis, it could be one of the contributing factors which could prevent endometriosis from developing into endometrial cancer.
Transformer-2 protein homolog beta (TRAB2B) is an mRNA splicing factor and functions in mRNA splicing and in transesterification in mRA binding496.
Serine/threonine-protein kinase N2 (PKN2) is a serine-threonine protein kinase family member496. It is an annexin and calmodulin protein class member. It has protein-kinase activity and is involved in calcium iron and calmodulin binding as well as calcium dependant phospholipid binding496,578. It is involved in calcium mediated signalling processes as well as protein amino acid phosphorylationas as well as involvement in the following three pathways: PDGF signalling pathway, Alzheimer’s disease amyloid secretase pathway and the muscarinic acetycholine receptor 1 and 3 signalling pathway526. The platlet-derived growth factor is one of the machrophage platlet-derived growth factors588 and the pathway is involved in
cellular growth and the process of angiogenesis. Studies showing the elevation of PDGF in the peritoneal fluid of endometriosis have partly attributed the proliferative properties of ectopic endometrial tissue forming endometriosis to this growth factor589. It too is being investigated as a potential novel therapeutic target589.
cGMP-dependent protein kinase 2 (PRKG2) is a non-receptor serine-threonine protein kinase member496,578. It binds proteins and has protein kinase and kinase regulator activity biologically involved in muscle contraction, intracellular signalling cascades, cellular mitosis and phosphorylation of amino acids. It is also involved in neurological system processes forming part of the cGMP dependent pathways and endothelin signalling pathway526.
Peroxisomal targeting signal 1 receptor (PEX5) targets proteins and is involved in peroxisomal transport as another membrane trafficking regulatory protein578.
Pituitary adenylate cyclase-activating polypeptide type I receptor (ADCYAP1R1) has G-protein coupled receptor activity and is seen in cellular stress responses496,578. It is a member of the antibacterial response proteins and is implicated in the cellular immune response. Modifications in this response might all contribute to the development of endometriosis in preventing aberrant cell elimination. Within the cell it has roles in cellular glucose homeostasis, synaptic and non-synaptic vesicle exocytosis enabling synaptic cellular transmission. It is also responsible for intracellular protein transport. Biologically it is involved in spermatogenesis, mesodermal and heart development and is involved in the G-protein coupled receptor protein signalling pathway and gonadotropin releasing hormone receptor pathway526. GnRH analogues are used for the enhancement of apoptosis in endometriotic cells590 and are one of the medical treatments used to suppress endometriosis pre-operatively or pre-fertility treatment. Understanding the effects of receptors such as ADCYAP1R1 on GnRH pathways could potentially provide another window to understanding this multifactorial disease.
Protocadherin-9 (PCDH9) binds calcium ion and has G-protein coupled receptor activity496,578. Biologically it has been linked to visual perception and the sensory perception of sound. At a cellular level, it is involved in cell-cell adhesion, cell motion and morphogenesis of the cellular components.
Embryonically it is involved in ectoderm and mesoderm development, nervous system development, heart and muscle organ development as well as the Wnt signalling pathways (Figure 5-14) and cadherin signalling pathways526 (Figure 5-15). The Wnt signalling pathway regulates cell growth motility and survival591 and molecular defects or alterations in this pathway have been associated with the development of multiple types of tumours591. Molecules such as PCDH9 might be responsible for minor changes within the function of this pathway which in endometriosis could result in ectopic cell survival or growth. Genes or proteins that are involved in cellular modification, signalling, motion and morphogenesis, all have a fine balance in controlling and ensuring correct cellular function and viability.
Any derangement or alteration in these molecules could potentially disrupt the normal cellular properties enabling a cell to grow, proliferate uncontrollably and potentially avoid or repress apoptosis. All these properties are seen in endometriotic cells and it is possible that it is through the understanding of the importance of molecules such as PCDH9 that we might obtain a better insight into development of the disease process of endometriosis and its associated pathways.
194 FIGURE 5-14
Wnt signalling pathways
526FIGURE 5-15
Cadherin signalling pathway526
Cytoplasmic dynein 1 light intermediate chain 2 (DYNC1LI2) binds proteins and is a structural component of the cytoskeleton496. Intracellularly, it is found within microtubule and acts as an enzyme modulator and has a role in the morphogenesis of cellular components and intracellular protein transport, RNA localisation and vesicle mediated transport. It has a role in the cell cycle and the intracellular signalling cascades. DYNC1LI2 is implicated in Huntington’s disease involving the dynein complex496,578.
Potassium voltage-gated channel subfamily C member 2 (KCNC2) also contains cation channel activity and is involved in cation transport. It is involved in cellular signal transduction and synaptic transmission.
Biologically it is involved in muscle contraction and neuronal action potential propagation496,578.
Golgin subfamily A member 8A (GOLGA8A) is another membrane trafficking protein. F-box/WD repeat-containing protein 1A (BTRC) is involved in Parkinson’s disease and three separate pathways:
hedgehog signalling pathway, Wnt signalling pathway and the toll pathway in drospophila526. The importance of the effects of the Wnt signalling pathway in tumorigenesis, cellular survival and proliferation has already been suggested previously591 with other molecules such as PCDH9 affecting the same pathway. Interestingly if multiple molecules affect this major pathway at different levels, this might further encourage cellular changes and endometriotic disease growth. There will be potential expressed counteracting or stabilising tumour suppressor genes such as LOXL1 which can explain the prevention of development of overt malignancy.
The following genes and proteins have been identified as downstream targets of hsa-mir-4274. Their roles and functions in literature are not currently clearly defined496,526,577,578
: Protein family with sequence similarity 3, member C1 (FAM3C), actin-binding rho-activating protein (ABRA), uncharacterized protein C6orf136 (C6orf136), MACRO domain-containing protein 2 (MACROD2). Bladder cancer-associated protein (BLCAP) may regulate cellular apoptosis and cell cycle proliferation by a mechanism independent from TP53/p53.
5.6.2.1 Summary of important identified points
From the above list of genes and proteins controlled by Hsa-mir-4274, one can see still see effects on the cellular functions of apoptosis, cell adhesion, migration, cytoskeletal structure, cellular signal transductions and vesicular formation. Various proteins controlled by this miRNA are also seen to potentially affect the immune system, altering ectopic cellular recognition, facilitating cellular evasion and enabling implantation and growth.
SP1 has a role in the immune system and in humans it is a constituent of the gonadotropin releasing hormone receptor pathway. Other identified molecules such as MAP2K6 and ADCYAP1R1 are also associated with effects on the gonadotropin releasing hormone receptor pathway. This is one of the targeted pathways by medical GnRH analogues that aim to promote apoptosis in endometriosis.
Other genes such as KLF3 are also know to affect B-cell mediated immunity which can be used by viruses such as EBV to invade proliferating cells which can evade immune recognition.
Molecules such as CREB1, MAP2K6 affect the RAS and MAPK pathways have been linked to hsa-mir-4274, all of which are associated with potential tumorigenesis, providing cells with the ability to proliferate, invade and grow. PCDH9 affects Wnt signalling pathways which also mediate cell survival and growth. These are all characteristics which are shared by endometriosis cells enabling ectopic growth.
Relating back to the information above, there are varied roles which each protein or gene is known to carry out though it is difficult to determine which of these pertains a leading role, or which of these follow on as a secondary response to alternate cellular processes.
It is beyond the scope of this chapter to determine which are the most likely proteins or genes or pathways to cause (or enable to a larger extent) the development of the disease of endometriosis. This would require a separate body of work with functional models to determine important molecular or protein roles.
Using the identified proteins, pathways and genes to perform functional modelling studies to establish which molecules have more pertinent roles that could serve as an interesting body for future work.