Literature reviewBreastcancer:Breastcancer is the most commonly diagnosed cancer and the top cause of cancer death amongfemales, approximately about 23% of the total cancer cases and 14% of thecancer deaths (Jemal et al., 2011). At 2012 a study performed in cancerincidence and mortality shows that breastcancer is the second most common cancer overall (1.7 million cases, 11.
9%) butranks 5th as cause of death (522,000, 6.4%) because of the relativelyfavourable prognosis (Ferlay et al., 2014).
In the year 2012, there were 1008cases of breast cancer in Jordan, of total breast cases 994 occurred in femalesand 14 cases in males (Al-Sayaideh et al., 2012).Thecausative agents that induce breast cancer are not clear but there are riskfactors that may influence or increase the possibility of getting it. Such as Micro-RNAmiR-21 which controls the expression of several genes that regulate tumorprogression, including RAB6A, a member of the RAS oncogene family, TGFb-inducedprotein, TGFb receptor II and Bcl2. Furthermore, recently it has been shownthat miRNA hsa-MiR-21 (miR-21) is up regulated in breast cancer andsignificantly associated with advanced stage at presentation and the increasedlevels of miR-21 are correlated with poor survival (Huang et al., 2009).
Epigeneticfactors and genes mutation also play role in breast cancer progression: silencingof the estrogen receptor and inactivation of BRCA1 and BRCA2 can occur throughmethylation. Gene mutation may involve dominant gene mutations such asmutations in BRCA1 and BRCA2 genes or SNPs (single nucleotide pleomorphism)which is change in a single nucleotide that result in increasing (MMP-1 andMMP-9 correlate well with advanced tumor stage) or decreasing (MMP-8 lead toenhanced breast cancer metastasis) genes expression (Tao et al., 2014). Risk ofdeveloping breast cancer is increased by early menarche, late menopause, and nulliparity;whereas, risk is reduced by higher parity and lactation. Furthermore, usingcombined hormone therapy after menopause increases breast cancer risk; the higherrisk appears to apply only to recent use. In addition, age, sex, and familyhistory consider important risk factors (Anderson, Schwab and Martinez, 2014).
Molecular classification of breast cancerfirst proposed by Perou and Sorlie, in there study they divided breast canceraccording to gene expression as follow: Luminal which differentiated in two orthree subgroups it reflect estrogen receptor (ER), ER regulatory genes and theexpression of genes expressed in normal luminal epithelial cells. Humanepidermal growth factor receptor (HER-2) positive it reflect HER-2 amplificationand overexpression. Basal breast cancer which reflect ER, progesterone (PR),and HER-2 negative and the expression of genes expressed in normal breast basalcells. A normal-like subgroup also been described (Perou et al., 2000). Thereare special types of tumor that does not fit Perou classification and itcomprise about 15-25% of breast cancer, according to ER expression they dividedinto two main groups: ER-positive group that consist of classic invasivelobular carcinoma, tubular, micropapillary, mucinous and neuroendocrine carcinomas.
The ER-negative group includes apocrine, pleomorphic invasive lobularcarcinoma, adenoid cystic, metaplastic and medullary carcinomas (Vuong et al.,2014). Table1: Histologic and molecular properties of specific type breastcancer (Eliyatkin et al., 2015) Molecular subtype Common histologic types HG ER status by IHC HER2 status by ISH/IHC Ki67 by IHC Specific IHC/molecular properties Luminal A Classical, lobular, tubular, cribriform 1 or 2 + – Low Luminal CK + E-cadherin +/- Luminal B Micropapillary 2 or 3 +/- -/+ High Luminal CK +, p53 mutations Basal-like Medullary, metaplastic, adenoid cystic, secretory 3 – – High Basal CK+, p53 mDNA repair loss, EGFR+/- mutations Molecular apocrine Apocrine, plemorphic lobular 2 or 3 – +/- High Androgene receptor+ Claudin low Metaplastic 3 – – High Cancer like stem cell, EMT like, low E-cadherin level HG: histologicalgrade, tumors are graded as 1, 2, 3, or 4, depending on the amount ofabnormality. G1:Well differentiated (low grade), G2: Moderately differentiated (intermediate grade). G3: Poorlydifferentiated (highgrade) (National Cancer Institute, 2018)IHC: immunohistochemistry +/- : mostlypositive, -/+ :mostly negative Triple-negativebreast cancer (TNBC):Thistype of cancers lack ER, PR, and HER2 amplification, treatment of these typecancers are difficult due to its heterogeneity and the absence of well-definedmolecular target (Carey et al.
, 2007). Using gene expression and clusteranalysis 6 TNBCs subtypes identified that show unique GE and ontologies, including2 basal-like (BL1 and BL2), an immunomodulatory, a mesenchymal, a mesenchymal stem–like, and aluminal androgen receptor (LAR) subtype. In this section, the focus will be onthe LAR because the MDA 453 belong to this subtype, this subtype is ERnegative, but gene ontologies are heavily enriched in hormonally regulatedpathways including steroid synthesis, porphyrin metabolism, and androgen/estrogen metabolism. Furthermore there are an increase in the expression of ARmRNA and numerous of downstream AR targets and coactivators (DHCR24, ALCAM,FASN, FKBP5, APOD, PIP, SPDEF, and CLDN8).
LAR subtype lack basal cytokeratin expression and express high levels ofluminal cytokeratins and other luminal markers (FOXA1 and XBP1),five cell lines matched to the LAR subtype (MDA-MB-453, SUM185PE, HCC2185,CAL-148, and MFM-223) (Lehmann et al., 2011). MDA-MB-453:Celllines representative of the LAR subtype (MDA-MB-453, SUM185PE, CAL-148, andMFM-223) express high levels of AR mRNA and protein. Furthermore they were moresensitive to bicalutamide than basal-like cell lines, also the LAR cell lineswere more sensitive to Hsp90 (which is chaperon required for AR proper folding)inhibitor 17-dimethylaminoethylamino-17- demethoxy-geldanamycin (17-DMAG)compared to basal-like and mesenchymal like cell lines.MDA-MB-453was transfected with AR targeting siRNA to investigate AR dependence on thiscell line, the knockdown verified at the mRNA and protein level, the ability ofMDA-MB-453 to form colonies was significantly reduced after knockdown of AR expressionas compared with control samples (Lehmann et al.
, 2011).MDA-MB-453cells harbor a K-RAS mutation at codon 13 (Gly 13 Asp GGC>GAC) which resultin constitutive activation of K-RAS also it has been confirmed that ERK1/2 ishighly phosphorylated in cells and it retain high ERK activity irrespective of FBSconcentration (VRANIC, GATALICA and WANG, 2011).Mutationin AR gene have been identified in MDA-MB-453, the resulting AR-Q865H varianthas compromised activity in response to DHT in relation to the wild type AR butit retains transcriptional responsiveness to DHT and it does not conferresponsiveness to non-androgenic ligands. It also possibly regulates a differentset of endogenous genes in relation to wild type AR (Moore et al., 2012). Epithelialmesenchymal transition (EMT):EMTprocess initially recognized during several stages of embryonic development andit has the ability to convert the epithelial cell to motile mesenchymal cell.
It is responsible of tissue remodeling events, including mesoderm formation,neural crest development, heart valve development and secondary palateformation etc. (Yang and Weinberg, 2008).EMT requiresmodifications in morphology, cellular architecture, adhesion, and movement capacity.Commonly used molecular markers for EMT include increased expression ofN-cadherin, vimentin, Fibronectin, Snail1 (Snail), Snail2 (Slug), Twist, Goosecoid,FOXC2, Sox10, MMP-2, MMP-3, MMP-9.
Nuclear localization of ?-catenin, Smad-2/3,Snail1 (Snail), Snail2 (Slug), and Twist that inhibit E-cadherin production. Furthermore,the cells morphology change into elongated mesenchymal shape and possessincreased migration capacity, the ability to invade, and scattering (Lee etal., 2006).EMTis induced by signaling pathways mediated by transforming growth factor ?(TGF-b) and bone morphogenetic protein (BMP), Wnt–?-catenin, Notch, Hedgehog,and receptor tyrosine kinases (Gonzalez and Medici, 2014). Transforminggrowth factor-? (TGF?) induces EMT through the formation of tetrameric complexof type I and type II receptors (T?RI and T?RII) to activate SMAD2 and SMAD3,which then bind with SMAD4. The SMAD complex translocates into the nucleus andcooperates with transcription regulators in the repression or activation oftarget genes.
TGF? can also induce non-SMAD signalling pathways through theactivation of PI3K–AKT–mammalian TOR complex 1 (mTORC1) signalling. Activationof Frizzled by Wnt ligands results in phosphorylation of low-densitylipoprotein receptor–related protein 6 (LRP6) by GSK-3? and the recruitment ofDishevelled (Dvl) and Axin to the plasma membrane enabling ?-catenin totranslocate to the nucleus, nuclear ?-catenin binds to members of the TCF/LEFfamily of transcription factors to promote EMT. Wnt-mediated induction of EMTthrough Snail2 by decreasing the expression of E-cadherin and increasing theexpression of fibronectin after the accumulation of ?-catenin in the nucleus (Lamouille,Xu and Derynck, 2014), (Gonzalez and Medici, 2014).Figure1: signaling pathways involved in EMT (Lamouille, Xu and Derynck, 2014)Roleof EMT in cancer:EMTprocess during cancer progression facilitate cancer cells invasion andmetastasis to distant sites, the role of EMT in oncogenesis achieved by theability of EMT transcriptional regulator to enhance cancer infiltration andmetastasis, such as SNAIL1 which correlate with increased aggressiveness andpoor survival in human breast cancer (Moody et al., 2005).
Therole of EMT is not confined to tumor invasion and metastasis only, it also playroles in cancer progression such as; resistance to cell death and senescence,resistance to chemotherapy and immunotherapy, evading immune system by inducingtolerance or modifying its phenotype and immunosuppression (Thiery et al.,2009). Figure2: Cellular cytoskeletons in epithelial?mesenchymaltransition (Zhang et al., 2017)Inbreast cancer, many transcription factors that regulate EMT have been found tobe activated in breast tumor resulting in EMT initiation and tumor progressionby forming a complex signaling network (Wang and Zhou, 2011). In breast cancer,partial or total loss of E?cadherin expression and increased N-cadherinexpression correlates with loss of differentiation characteristics,acquisition of invasiveness, increased metastatic ability (Liu et al.
, 2017).Cluster of differentiation CD44 (which is cell?surface protein that modulatescellular signaling) expression is upregulated in high?grade human breast cancers,and is associated with the level of the mesenchymal marker N?cadherin in these cancers(Brown et al., 2011). In normal non-invasive epithelial cells, ?-cateninlocated at the cell membrane binding to the cytoplasmic portion of E-cadherin,in EMT ?-catenin dissociate from cell membrane and located in cytoplasm thentranslocate in the nucleus to promote the transcription of genes that induceEMT (Zeisbergand Neilson, 2009). Furthermore, there are clinical evidences to support theupregulation of Wnt/? ?catenin signaling in invasive breast ductal carcinoma (Prasad et al., 2009).Increasedexpression of SLUG in invasive ductal carcinoma result in inducing EMT throughdownregulation of E-cadherin (Lopez et al.
, 2009), finally further studies onthe link between EMT markers and breast cancer will contribute to the identificationof biomarkers for early breast cancer metastasis prediction and to identify newtherapeutic targets in breast cancer (Liu et al., 2016).AndrogenReceptor (AR):Androgenreceptor belong to nuclear steroid hormone receptor (nuclear receptorsub-family 3, group C, gene 4) along with estrogen, progesterone,mineralocorticoid, and glucocorticoid receptors, it plays important roles inthe development and maintenance of the reproductive, musculoskeletal,cardiovascular, immune, neural and haemopoietic systems (Nuclear Receptors Nomenclature Committee,1999)( Davey et al.
, 2016). AR consists ofthree major functional domains: N-terminal domain (NTD), DNA binding domain(DBD) and C-terminal ligand binding domain (LBD) which connected to DBD by aflexible hinge region. The DBD is highly conserved among all steroid hormonenuclear receptors and it consist of two zinc fingers that recognize specificDNA consensus sequences (Heinlein and Chang, 2002).DBDenable direct DNA binding of AR to the promoter and enhancer regions ofAR-regulated genes, facilitating the activation functions of the N-terminal andligand binding domains to induce or repress the transcription of these genes,the highly conserved nature of DBD allow specific binding to the androgenresponsive element in genes promoter regions (Schoenmakers et al., 2000).
Theligand binding domain linked through hinge region to DBD and it mediates theinteraction between the AR and heat shock and chaperone proteins, also itfacilitate the interaction with the N-terminus of the AR to stabilize boundandrogens (Heinlein, 2002).Twotranscriptional activators have been identified: the ligand-independentactivation factor (AF-1), positioned in the N-terminal domain and required formaximal activity of the AR, and the ligand-dependent activation factor (AF-2),placed in the ligand binding domain responsible for making the coregulatorbinding site as well as enabling direct interactions between the N-terminal andligand binding domains (Wilson, 2011). Nuclear localization signal (NLS)located between the DBD and hinge region and its responsible for AR entrance intothe nucleus, while nuclear export signal (NES) responsible for exporting AR tothe cytoplasm upon ligand removal and it located in the ligand binding domain (Tanet al., 2014). Figure3: Functional domains of the androgen receptor (Davey et al., 2016).In DNAbinding-dependent actions of AR (canonical AR signalling), androgens bind tothe AR resulting in detachment of chaperone proteins, conformational change andexposure of NLS which induce nuclear localization of AR, where it dimerises andbinds to AR responsive elements within target genes promoters to enhance orrepress gene transcription in the presence of co-regulators. (van de Wijngaartet al.
, 2012). DNA binding independent actions of AR (non-canonical) in whichandrogen/AR complex activate second messenger pathways including ERK, Akt andMAPK, also indirect gene transrepression can happen, by the AR binding andsequestering transcription factors such as activator protein-1 (AP-1) (Lamontand Tindall, 2011). Roleof AR in EMT:ARreceptor can induce EMT through several mechanism, direct inhibition ofE-cadherin through the ability of AR to bind to potent repressive element inE-cadherin promoter after being activated by DHT, and induce morphologicalchanges from epithelial to mesenchymal-like appearance. Furthermore, clinicalsamples from invasive breast ductal carcinoma show high levels of AR andreduced E-cadherin expression (Liu et al., 2008).
ARcan induce EMT in prostate cancer through the activation of Snail zinc fingertranscription repressor resulting in significant migration and invasionpotential (Zhu and Kyprianou, 2010).Zincfinger E-box-binding protein 2 (ZEB2) which is one of the EMT transcriptionalmediator was found to be significantly upregulated after androgen stimulation,decrease in ZEB2 expression have been observed after AR silencing inandrogen-dependent prostate cancer cell line. This finding propose AR aspositive regulator of ZEB2 expression in androgen-dependent cells (Jacobet al.
, 2014). In prostate cancer activated AR can significantly increase theexpression of Slug even after 2hr from activation (Wu et al., 2012). Splice variants of AR contribute tocastration-resistant prostate cancer and EMT induction, AR splice variants 3(AR3) modulates the expression of TGF-?, IGF1, and several EMT associated genes(Khan et al., 2015). In triple negative breast cancer AR upregulate ZEB1(which is transcription factor involved in EMT) after being activated with DHT topromote EMT and cell migration (Graham et al.
, 2009). SLUG:Slugbelongs to Slug/Snail family of transcription factors, Proteins of this familyinclude Snail1, Slug/Snail2, Snail3/Smuc, and Scratch. They have a highlyconserved carboxy-terminal region that contain four to six C2H2-type zincfingers that bind to a subset of E box (CAGGTG) sites in the promoter regionsof genes that regulated by this family (Inukai et al., 1999). The N-terminalregion contains the SNAG transactivation domain, which is essential forSnail2-mediated repression and specific 28 amino-acid sequence (amino acids 96to 123) called the SLUG domain, both the N-terminal SNAG and the central SLUGdomains are required for efficient repression of the E-cadherin promoter.
Nuclear receptor co-repressor 1 (NCoR) interacts with Snail2through the SNAG domain, while C-Terminal Binding Protein 1 (CtBP1) is recruitedthrough the SLUG domain (Molina-Ortiz et al., 2012).Innormal development SLUG was first recognized in the neural crest, developingmesoderm and the limb of chick embryos and it shows dynamic pattern oftranscription during early development, furthermore knockdown of Slug gene usingantisense oligos result in specific developmental failure suggesting itsimportant role in embryo development (Nieto et al., 1994).
Slugdisplay increased expression during organogenesis, especially in proliferatingchondrocytes in bone of both neural crest and mesodermal origin, intestinal andstomach walls, craniofacial mesenchyme and mesenchyme of the lung and kidney(Oram et al., 2003).Studyperformed by (Shi et al., 2011)recognize Snail2 as a key regulator of the signals involved inmesodermal induction of neural crest, and its loss results in changes in theRNA levels of a number of BMP and Wnt agonists and antagonists.Polycombrepressive complex 2 (PRC2) consist of Eed, Ezh2 and Suz12 core component whichexpressed in neural crest cells and are required for neural crest markerexpression, during neural crest development slug and PRC2 cooperate with eachother in order to express the genes that associate with neural crestspecification and migration (Tien et al.