Telomere Length Regulation Through Epidermal Growth Factor Receptor .

using clinical studies, we and others have demonstratedthat mutations in the PI3K/AKT/mTOR signalingpathway also predict for resistance to cetuximab[18, 19].However, this remains clinically investigational becauseof conflicting data from some other groups[20, 21]. In acontinued search for biomarkers, our group has studiedtelomere length (TL) and its association with sensitivity/resistance to anti-EGFR therapy in CRC[16].We reported that TL has potential to be a noveland unique predictive biomarker of clinical outcome(progression free survival/PFS) to anti-EGFR therapy inpatients with KRAS WT metastatic CRC. There is extensiveliterature associating EGFR pathway with TL/telomerase.Our studies prove that longer TL corresponds to bettertherapeutic outcome in patients. Upon comparison,PFSs of other established therapeutic regimens for CRCoverlapped when separated into different cohorts basedon TL. This helped us to establish a unique interactionbetween TL and EGFR pathway (Figure 1). An addedstrength of this study was demonstration of hypothesis inin vitro and among patient samples. TL didn’t appear tobe a prognostic biomarker, but rather, a predictive one.The study, for first time, showed that malignant colonicepithelium corresponding to age-related decline in TLthat is widely observed in nonmalignant cells such asperipheral blood leukocytes as well as colonocytes. Thestudy also observed that when patients were categorizedas either localized disease (stages 1–3), or stage 4 atdiagnosis, there was a statistically significant difference inTL[16]. Cancer tissue in general has lower TL than normalmucosa, and there appears to be a positive correlationbetween TL and telomerase. Therefore, telomerase, theenzyme constitutively expressed in cancer, is unable toincrease TL in tumor cells beyond the adjoining normalmucosa[22].RNA template - telomerase RNA Component (TERC)and accessory components such as dyskerin (DKC1) enzyme complex that helps in extension of telomeres,TCAB1 (WRAP53) – localization protein, EST1A(SMG6) – protein aids in telomerase recruitment, andpontin and reptin (RUVBL1 and RUVBL2, respectively).TERT is the key molecule involved in regulation and ratelimiting step in enzymatic activity and carcinogenesis[26].Telomerase is required for the long-term proliferation ofhuman stem cells and cancer cells. There is mountingevidence for the existence of an important relationshipbetween telomeres and telomerase and cellular aging andcancer[27]. The mechanisms underlying TL maintenanceand telomerase expression involve transcriptional, posttranscriptional and epigenetic regulation[15, 28], and anin-depth understanding of these mechanisms may providenovel biomarkers and targets for early detection of disease,determination of disease prognosis, and the developmentof therapeutics.A large array of telomeric proteins regulates TLand telomerase activity (TA) and protects telomereends from being recognized as DNA breaks. Theseproteins together form the vast network of proteins atthe telomeres that ensure genome stability and integrity.The telomere interaction network is anchored by a sixprotein complex, also called telosome/shelterin, andis composed of telomeric repeat factor 1 (TRF1) and 2(TRF2), telomeric repeat-binding factor 1 [TERF1]interacting nuclear factor 2 (TIN2), tripeptidyl-peptidase1 (TPP1), protection of telomere 1 (POT1) and repressor/activator protein 1 (RAP1)[29]. Three of these proteinscan directly interact with telomere DNA - TRF1 and TRF2interact with the telomere duplex region, while POT1binds to telomere ssDNA. The N-terminal basic domainof TRF2, rich in glycine and arginine residues (GAR orbasic domain), can also bind to the non-coding telomericRNA [30]. Although TPP1 itself has no demonstrableDNA binding activity, it enhances the interaction of POT1with telomere ssDNA[31]. TPP1 can directly interactwith TERT and recruit the telomerase to telomeres[32].Maintenance of telomeres requires both DNA replicationby replication protein A (RPA), which binds to the ssDNA,and telomere ‘capping’ by POT1. Ablation of POT1leads to aberrant accumulation of RPA at telomeres andactivation of the ataxia telangiectasia and Rad3-relatedkinase [30]-mediated checkpoint response, suggesting thatPOT1 antagonizes RPA binding to telomeric ssDNA[33].RAP1 is recruited to telomeres through its interaction withTRF2, and TIN2 is recruited to by binding to TRF1[34,35]. TIN2 also functions as a bridge connecting TRF1/TRF2 with TPP1[34]. Subunits of shelterin also assembleother interacting proteins on telomeres. They bind todifferent regions of telomere and induce the formationof a T loop, a cap structure that deters DNA-damagesensing machinery from mistakenly repairing telomeres.The absence of shelterin causes telomere uncapping andTelomere, telomerase and structural complexitiesTelomeres are distinctive and repetitive nucleotidesequences found at the ends of linear chromosomes. Invertebrates, they consist of hundreds to thousands ofTTAGGG double-stranded DNA tandem repeat sequencesand terminate in single-stranded 3’-overhangs (singlestranded DNA/ssDNA) that invade into the doublestranded region to form the so-called telomere loop (Tloop) structure[23]. Among its main functions, telomeresmask double strand break DNA damage signals at theextreme of chromosome and prevent chromosomal fusionsand thus contribute to genomic stability. Telomeres shortenwith each round of cell division and this mechanismlimits proliferation of human cells to a finite numberof cell divisions by inducing replicative senescence,differentiation, or apoptosis[24]. Telomere shorteningcan act as a tumor suppressor too[25]. Telomerase, aribonucleoprotein structure that consists of the reversetranscriptase - telomerase reverse transcriptases (TERT),www.impactjournals.com/Genes&Cancer552Genes & Cancer

thereby activates damage-signaling pathways that maylead to non-homologous end joining (NHEJ), homologydirected repair, senescence, or apoptosis[36].In addition to telomere DNA and telomeric proteins,another factor in the telomere interaction network is RNA.Telomeres are also constitutively transcribed into telomericrepeat-containing RNAs or TERRA, which are long noncoding RNAs with variable length. TERRA localizes totelomeres and is transcribed from several subtelomericloci toward chromosome ends[37]. TERRA can interactwith a number of telomeric proteins (e.g. TRF1 and TRF2)and has been implicated in telomere heterochromatinmaintenance, telomerase regulation and telomere capping.They act as negative regulators of TL based on theirability to inhibit telomerase in vitro[15]. Downregulationof TERRAs occurs during cancer progression, a scenariothat requires the efficient elongation of short telomeres bytelomerase[38].Luo et al. in 2015 indicated that some componentsof telomere interactome can bind to telomere DNA orTERRA (directly or indirectly), suggesting that theseproteins may play an essential role in telomere regulation.For example, poly(ADP-ribose)polymerase 1 (PARP1), aninteracting protein of RAP1 and telomeric repeat-bindingfactor 2 (TERF2), functions as regulator of telomerelength and end protection, also binds to telomere DNAand TERRA[26]. PARP1 is capable of poly(ADP-ribosyl)ation of TERF2, which affects binding of TERF2 totelomere DNA[39]. Whether PARP1 regulates TERRAstability, localization and/or modification remains tobe investigated. Studies identified a RPA-to-POT1switch on telomeres that is orchestrated by TERRA andheterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).Integrated analysis of different datasets also revealedthat core telomeric proteins such as TRF2 can target togene loci of its interacting partners (e.g. PML), raisingthe possibility that TRF2 may regulate the dynamics ofprotein–protein interaction networks and the integrity oftelomeres through transcriptional control[40]. TERF2can recruit excision repair protein ERCC1 along withERCC4 (ERCC1/XPF complex) heterodimer to thetelomeric complex, which helps protect against telomererecombination with interstitial telomere-related sequences,and at the same time prevent NHEJ by blocking its accessto the G-strand overhang. TERF2 is also capable ofmodulating other DNA damage repair (DDR) proteinsor complexes such as MRE11A–RAD50–NBN (MRN),which detects presence of uncapped telomeres, Apollo,KU70 and UBR5, which is a chromatin regulator, totelomeres[41, 42]. Interestingly, a number of proteinsinvolved in signal transduction and metabolism appearto interact with core telomeric proteins and/or TERRA,thereby linking metabolic control and specific signalingpathways to telomere maintenance. For example, thesubunit of 3-methylcrotonyl-CoA carboxylase (MCCC2)interacts with TRF2 and TERRA. Mutations of MCCC2www.impactjournals.com/Genes&Cancerhave been shown to be associated with the autosomalrecessive disorder of leucine catabolism termed3-MethyIcrotonylglycinuria. The MCCC2–TRF2–TERRAinteraction may indicate telomere dysfunction in thismetabolic disease[37]. Recently, telomeric proteins havebeen found to play extra-telomeric roles in biologicalprocesses and diseases including NF-κB signaling, obesityregulation, NK cell immunity and neural tumor/stem cellfate control[37].Telomere length and EGFR pathwaysImmortalization and malignant transformationduring cancer involve a complex accumulation of geneticand epigenetic events mainly in the proto-oncogenes andtumor-suppressor genes, as well as the ability to maintaintelomeres[14]. TA is observed in almost 90% of humancancers and immortalized cells but not in normal tissuesof somatic origin and thus is a critical step for multistepcarcinogenesis. In addition to elongating telomeres anddeducing an immortalized state, telomerase has other rolesin tumor progression[43]. The role of telomere dysfunctionin colorectal carcinogenesis is still largely undefined.Several studies demonstrated that telomeres were shorterin CRCs than in adjacent normal mucosa[44-47], butthis finding was not confirmed by other studies[48, 49].Growth signals are directly or indirectly involved intelomerase regulation[28]. EGFR overexpression can leadto malignant transformation and activation of telomerasevia survival pathways such as PI3K/AKT/mTOR, MEK/ERK 1/2 and JAK/STAT[50]. Upon activation, variousadaptor and effector molecules such as SHC, GRB2-7,c-CRK-p38, PLC-γ, SHP1,2, CBL downstream of EGFRhelp to link to these pathways[51]. A more thoroughunderstanding of telomerase regulation may provide notonly a molecular basis of cancer progression but also as away to manipulate TA as a potential therapeutic modality.EGF is a representative growth factor that facilitatesproliferation of a variety of cell types. Once EGFRpositive cells are exposed to EGF, TA is upregulatedfollowing activation of hTERT mRNA expression.This is a rapid effect, observed within 6 hours aftertreatment. No requirement for de novo protein synthesiswas observed, suggesting a direct effect of EGF[43].There are several lines of evidence that specific signaltransduction pathways mediate this regulation. A specificMEK inhibitor of the RAS/MEK/ERK pathway abrogatesEGF-induced activation of hTERT. Transactivation ofhTERT by EGF requires a specific promoter element(TTCCTTTCCG) located at -22, a consensus bindingmotif for ETS-2 proteins, known to be the major target ofEGFR signaling[43]. Two highly recurrent mutations attwo sites within core promoter region of hTERT generatea consensus binding motif for ETS-2, which functionsas transcriptional repressor, activator or both to regulateexpression of telomerase[15]. Signaling molecules553Genes & Cancer

activated by the EGFR, including ERK, SRC, and AKTalso have regulatory role on TA. These findings suggestthat EGF signals utilize the RAS/MEK/ERK pathway toactivate hTERT expression[52].Another line of study suggests deregulated EGFRpathway induces TA via PI3K/AKT-mediated directphosphorylation and hypoxia-inducible factor-1α (HIF1α)-mediated transcriptional regulation of hTERT incancer. Ionizing radiation increases TA in various cancersby a posttranslational mechanism implicating PI3K/AKT pathway, and is inhibited by PTEN [53, 54]. Yanget al., 2008, suggests hTERT expression is regulated bylysophophatidic acid via PI3K pathway and transcriptionfactor, HIF-1α[53]. Two phosphorylation sites ofAKT within hTERT protein help for AKT-dependentphosphorylation and subsequent activation of hTERT.Activated hTERT eventually shuttles from the cytosolto the nucleus[53]. In another study, phosphorylationof hTERT by PKC isoenzymes has been identified asan important mechanism of telomerase regulation[55].Upregulation of EGFR expression and activation ofMAPK signaling pathway were observed in hTERTimmortalized nasopharyngeal epithelial cells. Lack of TAunder hypoxic conditions and in the presence of a MAPKkinase 1-specific inhibitor in solid tumors suggests theimportance of MAPK pathway in regulating telomeraseunder hypoxia[56]. The MAPK pathway is involved inthe activation of HIF1 expression, and HIF1 has beenshown to bind to the promoter region of hTERT, likelyat the 1 hypoxia response element site in TERT whichis overlapping E-boxes, and regulate TA that was provenby chromatin immunoprecipitation assay and TRAPassay, respectively[57]. HIF-1α binding site within thehTERT core promoter seems to play an important role inthe transcriptional regulation or transactivation of hTERTin EGFR overexpressing cells[14]. HIF-1α has beenidentified as a positive regulator of telomerase expressionin different normal and tumor cell lines. Moreover, HIF-1αhas been characterized as a downstream target of EGFRand HER2 signaling via the PI3K/AKT pathway andMAPK. Activation and stabilization of HIF-1α are doneby phosphorylated AKT and subsequent phosphorylationof HIF-1α protein[50].to these E-boxes through heterodimer formation withMAX proteins and activates transcription of hTERT.This is a direct effect of MYC that does not require denovo protein synthesis. MAD proteins are antagonistsof MYC and switch from MYC/MAX binding to MAD/MAX binding decreasing promoter activity of hTERT[61].Overexpression of c-MYC is frequently observed ina wide variety of tumor types, and usually results fromchromosome translocation involving the c-MYC genesin addition to gene amplification[62]. It is possible thatHIF-1α binding is affected by competition with thesefactors. Hypoxia downregulates c-MYC expressionor facilitates degradation of c-MYC and binding withhTERT[63]. During the competition for hTERT promoterbinding between HIF-1α and c-MYC, if there’s hypoxia,the promoter is predominantly bound by HIF-1α[57].Transcriptional derepression of TERT via hyperactivationof MYC might be the rationale for telomere-independentfunctions of telomerase which include regulation ofmitochondrial activity, cell proliferation and apoptosis,WNT/β-catenin signaling, NF-κB signaling, and DDR,all of which may play roles in oncogenesis[64]. MYCdriven oncogenesis is regulated by telomerase. Althoughoverexpression of the c-MYC gene is observed in asignificant proportion of tumors, some tumors lackMYC overexpression despite the presence of TA. Uponactivation, EGFR drives the phosphorylation/activation ofseveral signal transduction pathways and transcriptionallyregulated pathways including NF-κB. Activation of NFκB promotes tumor progression processes includingproliferation, maturation, growth, angiogenesis,invasion, metastasis, clonal expansion and inhibitionof apoptosis[65]. RAS/MEK/ERK signaling pathwaysmay be important for hormone/estrogen-mediatedtranscriptional regulation of hTERT[66]. EGF-activatedhTERT expression is mediated via the MEK pathway andtranscription factor ETS-2 that targets hTERT promoter inlung cancer cells[13].MYC and Wnt signaling pathways are keyimportance for cancer and stem cell biology. TERTis proven to be transcriptionally regulating Wnt–βcatenin signaling pathway and has RNA-dependentRNA polymerase activity when in a complex withRNA component of mitochondrial RNA processingendoribonuclease[67]. β-catenin regulates TERTexpression through the interaction with KLF4, acore transcription factor belongs to large SP1-liketranscription factor family and important componentfor pluripotency[68]. TERT functions as a cofactor inβ-catenin transcriptional complex through interactionswith BRG1 (also known as SMARCA4), which is aSWI/SNF-related chromatin remodeling protein[69].EGFR-mediated MAPK signaling attenuates Grouchomediated gene repression, establishing a node for crosstalkbetween the EGFR, Notch, WNT, and TGF-β signalingpathways[70]. Growth factor and cytokine receptorsTelomere length, MYC and Wnt signalingEGFR/MEK/ERK/IKK/mTORC1 is the keyupstream pathway of NF-κB activation[58]. FunctionalNF-κB mediates TA by binding to the κB binding regionin the promoter region of hTERT[59]. Constitutiveactivation of NF-κB signaling also leads to upregulationof other transcription factors such as MYC, MAD andETS-2, and genes such as IL-6 and BMI-1, and inducingresistance to chemotherapy and radiation and proliferatingcancer[60]. In the core promoter-200-bp proximal regionof hTERT, multiple E-boxes are located. MYC bindswww.impactjournals.com/Genes&Cancer554Genes & Cancer

stimulation induces transcriptional and posttranslationalactivation of hTERT through JAK/STAT pathwayand JAK/PI3K/AKT/HSP90/mTORC1 pathway inhematological malignancies[71, 72].activation and maintenance, and often associated withgrowth factor receptor gene amplification in cancers[79,80]. Nguyen et al. (2013), states that, in brain cancer, ALTphenotype is associated with ATRX mutations and theprotein loss is linked with expressions of IDH1 mutantprotein and p53, and absence of EGFR amplification[81].While EGFR genetic polymorphisms being risk factor,DAXX mutations are proved to be limited to onlypediatric gliomas[82]. Many molecular details of the ALTmechanism remain unknown, but it has been proposed thatvarious HR proteins are involved.Telomere length and cell cycle regulatorsPrimary/somatic cells divide exponentially makingtelomeres shorten from 15 kilobases (kb) until theyreach a critical length, 4–6 kb, which induces cell-cyclearrest, monitored by p53 and RB1 and leads to massivegenomic instability and cell death via apoptosis orreplicative senescence. TA before senescence allowssomatic cells to divide indefinitely and maintain a stablegenome through a process called immortalization. TAbefore erosion is complete rescues the genome frominstability by re-establishing telomere maintenance. TAafter the accumulation of mutations results in an unstablegenome, allowing transformed clones that carry multiplemutations to become immortal and oncogenic[73].Telomere shortening inhibits tumorigenesis in modelswith intact p53 pathways. EGFR overexpression andmutations in p53 contribute to epithelial to mesenchymaltransition (EMT) in TA-immortalized esophageal cellsduring carcinogenesis[74]. EGFR overexpression triggersoncogene-induced senescence, accompanied by inductionof cyclin dependent kinase inhibitors p15INK4B, p16INK4Aand p21[75]. EGFR-induced cell cycle progressionand proliferation correlated with the phosphorylationand cytoplasmic translocation of p21. On the contrary,overexpression of p21 leads to inhibition of both TAand hTERT mRNA expression in progesterone-positivecells[76]. More than 48 hours of exposure to progesteronepromoted cyclin-dependent kinase inhibitor p21/WAF1/CIP1-mediated inhibition of estrogen-induced activationof hTERT mRNA expression[77]. It is unclear how p21inhibits TA in presence of progesterone, but it is likelyattributable to indirect action through cell cycle arrest[28].SUMMARYIn summary, TL is managed globally by telomeraseenzyme, which is a multi-component complex. TL is alsocontrolled by multi-protein shelterin units at their tips.TA occurs through EGFR-mediated pathways and mainlyexecuted by PI3K/AKT/mTOR, RAS/RAF/MEK/MAPKand JAK/STAT signaling. PI3K and MAPK pathwaysalso regulate expression of transcription factor HIF1α, which is a positive transactivator of hTERT. hTERTcore promoter binding is dependent on EGFR-mediatedactivation of NF-κB through MAPK/mTORC1 pathwayand expression of other transcription factors such asMYC, MAD, MAX and ETS-2. MAPK signaling alsoestablishes node for crosstalk between EGFR, Notch,Wnt, and TGF-β signaling pathways. EGFR regulates TAvia shortening of TL beyond threshold leading to p53 andp21 induced cell cycle arrest. Homologous recombinationbased alternative lengthening of telomeres is independentof TA. ALT mechanism is controlled by ATRX and DAXXgenes on which EGFR plays a role.CONFLICTS OF INTERESTThe authors declare no conflict of interest.REFERENCESAlternative lengthening of telomeres and EGFRA subset of cancer/immortalized cells maintain TLfor hundreds of population doublings in the absence ofTA, and it was therefore deduced that they must have analternative lengthening of telomeres (ALT) mechanismprobably involving genetic [homologous] recombination(HR). Telomerase uses an RNA template for de novosynthesis of telomeric DNA, whereas ALT involvessynthesis of new telomeric DNA from a DNA templatevia HR[78]. The template may be the telomere of anotherchromosome or another region of the same telomere viat-loop formation or sister telomere recombination[78].Loss of function or mutations on alpha thalassemia/mental retardation syndrome X-linked (ATRX) or deathdomain associated protein (DAXX) genes leads to ALTwww.impactjournals.com/Genes&Cancer5551.Arshad HM, Tetangco E, Shah N, Kabir C, Raddawi H.Racial Disparities in Colorectal Carcinoma Incidence,Severity and Survival Times Over 10 Years: A RetrospectiveSingle Center Study. J Clin Med Res. 2016; 8: 777-786.2.Armaghany T, Wilson JD, Chu Q, Mills G. Geneticalterations in colorectal cancer. Gastrointest Cancer Res.2012; 5: 19-27.3.Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CACancer J Clin. 2017; 67: 7-30.4.Shivakumar BM, Chakrabarty S, Rotti H, Seenappa V,Rao L, Geetha V, Tantry BV, Kini H, Dharamsi R, Pai CG,Satyamoorthy K. Comparative analysis of copy numbervariations in ulcerative colitis associated and sporadiccolorectal neoplasia. BMC Cancer. 2016; 16: 271.5.Curtin K, Slattery ML, Samowitz WS. CpG islandGenes & Cancer

methylation in colorectal cancer: past, present and future.Patholog Res Int. 2011; 2011: 902674.6.Muller MF, Ibrahim AE, Arends MJ. Molecular pathologicalclassification of colorectal cancer. Virchows Arch. 2016;469: 125-34.7.Worthley DL, Leggett BA. Colorectal cancer: molecularfeatures and clinical opportunities. Clin Biochem Rev.2010; 31: 31-8.8.Oikonomou E, Koustas E, Goulielmaki M, Pintzas A.BRAF vs RAS oncogenes: are mutations of the samepathway equal? Differential signalling and therapeuticimplications. Oncotarget. 2014; 5: 11752-77.9.17. Wormald S, Milla L, O’Connor L. Association of candidatesingle nucleotide polymorphisms with somatic mutation ofthe epidermal growth factor receptor pathway. BMC MedGenomics. 2013; 6: 43.18. Hong S, Kim S, Kim HY, Kang M, Jang HH, Lee WS.Targeting the PI3K signaling pathway in KRAS mutantcolon cancer. Cancer Med. 2016; 5: 248-55.19. Wang XW, Zhang YJ. Targeting mTOR network incolorectal cancer therapy. World J Gastroenterol. 2014; 20:4178-88.20. Bardelli A, Siena S. Molecular mechanisms of resistanceto cetuximab and panitumumab in colorectal cancer. J ClinOncol. 2010; 28: 1254-61.Van Krieken JH, Rouleau E, Ligten

or less often (20-30%) BRAF mutations and are MSS or MSI-low[7]. The KRAS and NRAS mutations are activating oncogenic mutations at codons 12, 13 and 61, and the BRAF mutation is the classical V600E activating mutation. Mutations in KRAS or BRAF lead to hyperactivation of MAP Kinase and PI3K pathways[8]. RAS signaling pathway being downstream .