| Background and Significance: Part 1 Treatment of non-small cell lung cancerLung cancer is one of the world's most common and deadly types of tumor.Non-small cell lung cancer(NSCLC)is a highly heterogeneous kind of tumor with a number of different cells of origins,accounting for more than 85% of newly diagnosed lung cancer incidences(1).There are a number of methods that have been used in the treatment of NSCLC,and one of the most important treatments is apoptosis-inducing chemotherapeutic agents such as didiammine platinum(II)(commonly referred to as cisplatin,CP).CP is used to treat a variety of malignant solid tumors,including testicular cancer,ovarian cancer,head and neck cancer,colorectal cancer,bladder cancer and lung cancer(2).CP eliminates tumor cells via different mechanisms,but the most important is the induction of DNA damage,followed by activation of DNA damage response and induction of mitochondrial apoptosis.Compared with normal cells,DNA damage is more likely to occur in rapidly proliferating tumor cells.Therefore,the use of cisplatin in the treatment of multiple tumors,including NSCLC,has achieved some clinical results,effectively prolonging the survival of patients with NSCLC(3).However,although patients receiving cisplatin treatment tend to have a good initial response,the treatment often leads to resistance and treatment failure(3).The study of cisplatin resistance is currently one of the major focuses of NSCLC drug development and targeted therapy,and these studies have found that cisplatin resistance involves several aspects that can be roughly divided into four categories(4,5).First,the abnormal activity of DNA damage/repair protein in tumor cells;second,failure of drug uptake by the tumor;third,intracellular mechanisms leading to drug inactivation of failure to reach the DNA targets;Fourth,the induction of anti-apoptotic proteins.It is worth noting that cisplatin resistance may have a variety of mechanisms,which may vary depending on the cell types.Therefore,regardless of how the mechanism of resistance is explained,cisplatin-based combinatorial therapy with other agents,aiming to improve the susceptibility of cancer to cisplatin,has become the main goals for chemotherapy.Recent advanced chemotherapy strategies include a multi-drug combination,coupled with identification of personalized histology/genetic testing at the molecular level,leading to the approach that different groups of patients are able to take different strategies to maximize the role of cisplatin(6-8).However,in some clinical trials,combination therapy still failed to control early(3-5 weeks)recurrence(3).Therefore,the study of the treatment of cisplatin-tolerant NSCLC cell subsets will be necessary to help us understand and reverse the resistance of these cells,which would lead to the future treatment of NSCLC.Part 2 Extra-telomeric function of telomeric binding proteinsStudies over the past two decades have shown that telomere dysfunction is critical to the development of tumors(9,10).Telomere dysfunction includes telomere sequence loss,telomere structure abnormalities and telomerase abnormalities,with its role in tissue aging and tissue regeneration to be the focus of current research.On the other hand,some of the factors that have been shown to be telomere-binding proteins in previous studies have also been found to have binding sites at different locations away from telomere.More importantly,some of these factors function independent of telomeres(11).The extra-telomeric function of telomeric binding proteins is found to be associated with a variety of molecular biological phenomena such as mitochondrial function,inflammation,embryonic development,gene expression regulation,metabolism,stem cell stability,cell adhesion and cancer(12,13).The current research focuses on the extra-telomeric functions of telomere-binding protein complexes named telterin,as well as their co-factors(14).These studies will enhance our understanding of identifying the link between telomere structure and other molecular biology processes,providing valuable information and help explain the pathogenesis of human genetic diseases,aging and cancer,and thus contribute to the understanding of human health and disease.An important example is the mammalian RAP1 gene(repressor/activator protein-1,repressor/activator protein-1;also named TRF2 IP,telomeric repeat binding factor-1,telomeric repeat binding factor 2 interacting protein-1)(15).The RAP1 gene of mammals is homologous to that of budding yeast Rap1.In yeast,Rap1 is the main binding protein of yeast telomeres and plays an important role in controlling telomere structure.It functions through protecting the telomere,recruiting related factors and telomerase to control telomere length as well as function(16-18).In addition,yeast Rap1 also has extra-telomeric function as a transcription factor and controls the survival and proliferation of yeast cells by controlling the expression of glycolytic enzymes and ribosome-related genes(19).On the other hand,the detailed interaction between Rap1 and telomeres is not very well understood.One theory suggests that it binds directly to telomere DNA,while another theory suggests that RAP1 is thought to bind with telomere through a mediator TRF2(20,21).Recent studies are also attempting to elucidate the role of Rap1 gene in mouse development.At present,two different Rap1 knockout mice models have successfully deleted exon 2 and exon 3 of Rap1 through the Cre-LoxP homologous recombination system(22,23).However,one of the studies reported that RAP1 knockdown resulted in embryonic development during death from 6.5 days to 45 days(23);in another case Rap1 knockout was found to be dispensable for mice survival and fertility(22).Consistent with the later discovery,some other studies have found that the absence of Rap1 in epithelial cells is not fatal through tissue-specific knockout mice,but leads to premature onset of skin pigmentation and some of the aging and obese phenotypes,especially in female animals(22).These seemingly contradictory results illustrate the importance of clarifying the function of RAP1 in mammals and especially in human disease.Part 3 Extra-telomeic function of RAP1 gene in mammalsAlthough the results from the study of Rap1 transgenic mice have been controversial,studies in mammalian cells and tissues did reveal that the function of Rap1 is independent of telomere.Martinez et al.Found an extra-telomeric binding site of Rap1 by using chromatin immunoprecipitation sequencing(Ch IP-seq),and further found that these sites have a shared tandem sequence of(TTAGGG)(11).These extra-telomeric Rap1 binding sites are enriched in the sub-telomere region,and the genes encoded by the DNA sequences in these regions have different extents of down-regulation upon Rap1 deficiency(11).Similarly,the role of RAP1 as a non-telomeric-dependent transcription factor can be amplified by some specific enhancer sequences.In addition,Rap1 also binds to the chromosomes of the non-gene coding region,and,these binding sites also share the(TTAGGG)tandem repeats(24).The effect of Rap1 binding in these regions may include stabilizing the genome and reducing the generation of recombination in these regions(24).Biochemical studies have shown that mammalian Rap1 has lost part of the DNA binding domain compared to yeast Rap1 protein,a phenomenon that may help explain that Rap1 is not essential for telomere protection of mammalian cells(20)The At the same time,however,Rap1 was also found to interact with factors other than TRF2 to aid in gene transcriptional regulation(15).Gene analysis showed that down-regulation of Rap1 knockdown in mouse embryonic fibroblasts involved multiple signaling pathways,including signals that promote cell proliferation,cell adhesion molecules,metabolic enzymes,insulin synthesis-related genes,peroxides Peroxisome proliferatoractivated receptors(PPAR)signaling molecules and growth hormone effect molecules(17).On the other hand,the ABC transporter and the genes involved in type 2 diabetes were significantly upregulated after Rap1 knockout(17).Therefore,the lack of Rap1 can affect multiple biological processes.More importantly,human RAP1 has also been identified with its extra-telomeric function(25).Through the whole genome functional screening,RAP1 was found among the important mediators of the NF-?B signaling pathway.In multiple human cells,it was found that overexpression of RAP1 can induce NF-?B signal,and RAP1 deficiency will inhibit NF-?B activity(25).In these cells,RAP1 expression is located not only in telomeres,and not even in the nucleus;part of the RAP1 molecule was found in the cytoplasm,becoming the component of some macromolecular complexes.The results of immunoprecipitation showed that RAP1 binds directly to the inhibitory NF-?B kinase(IKKA-IKKB-IKKG),which is necessary for the phosphorylation of the p65 subunit in NF-?B,allowing NF-?B to translocate to the nuclei and recruit the necessary chromatin remodeling proteins for transcriptional activation of the target gene.The interaction between RAP1 and IKK stabilizes this complex,allowing it to mediate phosphorylation and nuclear translocation of p65,which allows transcription of NF-?B downstream genes to be activated.This finding provides another perspective for the role of RAP1 in transcriptional regulation.Interestingly,the relationship between the two factors forms a positive feedback loop: the expression level of RAP1 is regulated by NF-?B signaling while RAP1 activates NF-?B signaling pathway.At present,two NF-?B binding sites have been found in the promoter region of the RAP1 gene(25).Thus,RAP1 in the cytoplasm is an important co-activator of the NF-?B signaling pathway(25).Except for TRF2,no new molecules were found to be able to bind to RAP1,especially to mediate its extra-telomeric function.However,the function of RAP1 was identified to be independent of telomeres.More importantly,the different expression of RAP1 in the nucleus and cytoplasm provides the possibility that it affects molecular and cellular process through different mechanisms,and this distribution also allows RAP1 to be subject to multiple layers of regulation(16,26).The amount of TRF2 that is associated with telomere can vary depending on cell type,differentiation,embryonic development,and age(23).The nontelomere-binding RAP1 may be a way to regulate the transcriptional network by controlling several biological processes,such as inflammatory responses and metabolism(17).As an important mechanism to control cell senescence,RAP1 levels(both telomere binding and non-telomere binding)may decrease with aging,thereby affecting gene expression changes,resulting in aging-related phenotypes.In particular,the disorder of the NF-?B pathway has been associated with human disease,particularly cancer(27).Constitutive activation of NF-?B signaling has been shown to contribute to the development and progression of various malignancies such as lung cancer,breast cancer,colon cancer and so on.Because of the activation of RAP1 for the signal pathway,its extra-telomeric function is thought to contribute to tumorgenesis.In this regard,Teo et al.found that the level of RAP1 in tumor cells was significantly higher than that of adjacent normal cells.Meanwhile,the positive correlation between the malignant levels of tumor expression levels was also identified in breast cancer(25).However,the detailed mechanism of RAP1 mediating malignancy,especially the activation of p65 in these tumors and the dependence of such activation on RAP1,remains to be resolved.Part 4 Focus of this studyThe results of the studies listed above indicate that extra-telomeric function of RAP1 is essential during multiple biological processes,through the regulation at both transcriptional and protein level for different signal pathways.In particular,RAP1 is a necessary and important regulatory factor for NF-?B signaling,which plays a central role in the development and progression of lung cancer.The constitutively activated NF-?B signal promotes proliferation of NSCLC cells,more importantly,mediates its resistance to chemotherapeutic agents such as cisplatin.However,it remains unclear about the function of the RAP1 in NSCLC.Therefore,we proposed this study to identify the role of RAP1 in NSCLC progression,specifically focusing on the correlation between RAP1 and cisplatin resistance as well as therapeutic outcome.In this study,we found that:(A)the expression of RAP1 in the cytoplasm of NSCLC was positively correlated with the degree of malignancy;(B)cytoplasmic RAP1 was overexpressed in NSCLC compared with normal lung tissue;(C)RAP1 promotes the proliferation of NSCLC cells and mediate cisplatin resistance by upregulating NF-?B-dependent anti-apoptotic protein BCL-2;(D)RAP1 deficiency renders NSCLC cells sensitive to cisplatin;(E)RAP1 is crucial for the survival of cisplatin-resistant NSCLC cells.Our results suggest that RAP1 contributes to tumor progression and cisplatin resistance,suggesting that RAP1 can be a therapeutic target for cisplatin-resistant NSCLC.Results Summary:Part 1 Expression of cytoplasmic RAP1 is associated with malignancy in NSCLC patientsPrevious studies have shown that cytoplasmic RAP1 was detected in breast cancer tissue,the pathology scores of which were positively correlated with the tumor grades(25).To test the hypothesis that cytoplasmic RAP1 is a biomarker for higher-grade NSCLC,we immunostained for RAP1 and quantified its cytoplasmic and nuclear expression in 93 lung adenocarcinoma and 75 lung squamous cell carcinoma tissues.The peri-tumoral normal tissue was used to reflect RAP1 expression in non-malignant cells.Both cytoplasmic and nuclear RAP1 were increased in NSCLC tumors compared with normal tissues,but the difference in cytoplasmic RAP1 appears to be greater.A higher level of cytoplasmic RAP1 expression was associated with a higher grade of NSCLCs.Moreover,higher cytoplasmic RAP1 expression was associated with poorer prognosis of adenocarcinoma patients.These analyses identify cytoplasmic RAP1 as an indicator of high-grade NSCLC,suggesting that it may play a critical role in cancer progression.Part 2 RAP1 is required for the growth of NSCLC cellsTo test the hypothesis that RAP1 is enriched in the cytoplasm of NSCLC cells,we compared the expression of RAP1 between three NSCLC cell lines and two lung epithelial cell lines.The NSCLC cells tested here include adenocarcinoma A549 and PC9,together with squamous cell carcinoma HCC827,while we were also using the normal cells including bronchial epithelial cells 16 HBE and small airway epithelial cells HSAEC1-KT.RAP1 expression was enriched especially in the cytoplasmic fraction of NSCLC cells,which also indicates that cytoplasmic RAP1 will facilitate malignancy.Due to the challenge to specifically inhibit cytoplasmic RAP1,we tested the alternative hypothesis that RAP1 mediates NSCLC cell proliferation.Therefore,lentiviruses encoding either one of the two short hairpin(sh)-RNA against RAP1(shRAP1-1 and shRAP1-2)or a scramble shRNA were applied to three different NSCLC cells lines.RAP1-deleted NSCLC cells exhibited defect in cell viability and growth over culture,as shown by both CCK-8 cell viability assay and colony formation assay.These results demonstrate that RAP1 is required for the growth of A549 cells.Part 3 RAP1 induces NF-?B signaling in NSCLC cellsDue to the limitation of technique,we are not able to study the function of cytoplasmic RAP1 by exclusively deleting it in the cytoplasmic compartment.However,RAP1 per se is insufficient for telomere protection(22),suggesting that cytoplasmic RAP1 is the main contributor to induce cell proliferation as seen above.We next detected the effect of RAP1 deletion on the activity of NF-?B signaling.Previous studies have demonstrated that cytoplasmic RAP1 acts as IKK adaptor proteins which mediate the phosphorylation of p65(25).Indeed,when RAP1 was deleted in A549 cells,we observed a decrease of phosphorylated p65 in the nuclear fraction.Phosphorylation of I?B?,which promotes NF-?B signaling,was also decreased with the deletion of RAP1.Further,the transcription of IL-1,MCP-1 and CD44,which are known to be positively regulated by activated NF-?B,was decreased after knocking-down RAP1.Together,these results suggest that RAP1 mediated NF-?B activation in NSCLC cells,which might be the mainly pathway by which RAP1 mediates NSCLC cell growth.Part 4 RAP1 mediates cisplatin resistance in NSCLC cellsActivated NF-?B is known to render the cancer cells resistant to chemotherapy(27,28).Moreover,CD44,whose transcription is regulated through NF-?B signaling,was discovered to be enriched in a cisplatin(CP)-resistant population(29).Therefore,we tested the hypothesis that RAP1 suppresses the sensitivity of NSCLC cells to CP.First,the RAP1 shRAP1-or scramble shRNA-transduced A549 cells were treated with different concentrations of CP for 48 h.A relative high dose of CP(>2?M)was found to eliminate almost all cells in both groups.Nevertheless,when less CP was applied,RAP1-deleted cells displayed worse survival compared to the scramble shRNA transduced cells.Unexpectedly,over-expressed RAP1 only modestly increase A549 cell growth;however,a larger amount of CP was required to eliminate RAP1-overexpressed cells at an extent similar to that achievable with the control cells.We next performed a competition assay in which normal A549 cells(GFP-)were mixed with RAP1-overexpressing or deleted cells(GFP+)at a 1:1 ratio and treated with 2?M of CP,harvested at 24 h and 72 h,and analyzed for the percentage of GFP+ cells.When treated with CP over time,the cells harboring overexpressed RAP1 became the dominant population while RAP1-deficient cells got rapidly eliminated.These results indicate that RAP1 expression has a positive correlation with CP resistance.CP acts through generating DNA damage in the proliferative cancer cells,which ultimately leads to their apoptosis.However,RAP1 does not seem to have significant impact on CP induction of DNA damage per se.Hence,our hypothesis is that RAP1 might inhibit apoptosis triggered by the DNA damage response.To prove this,we treated RAP1-knockdown,RAP1-overexpressing and normal A549 cells with CP,and analyzed for the expression of cleaved caspase-3(C-Cas3)and BCL-2.RAP1 overexpressed cells displayed a similar baseline extent of apoptosis compared to normal cells,but CP did not trigger an obvious upregulation of cleaved Caspase-3;in contrast,CP facilitated the RAP1-deleted cells to express high level of cleaved Caspase-3 at an early time point during the treatment.Similarly,the proportion of Annexin V+ apoptotic cells after CP treatment was decreased in RAP1-overexpressing cells and conversely,increased in RAP1-deleted cells.When examining the anti-apoptosis factor BCL-2,we discovered a positive correlation between RAP1 and BCL-2 expression even without CP treatment.In normal and RAP1-overexpressing cells,CP treatment slightly induced BCL-2 expression which might be a negative feedback of facilitated apoptosis.However,the CP-treated,RAP1-deleted cells showed no BCL-2 up-regulation compared with untreated cells,suggesting that RAP1 is necessary for BCL-2 induction in response to CP.Thus we would conclude that RAP1 inhibits CP-induced apoptosis to mediate CP-resistance.Part 5 Cisplatin resistance is associated with RAP1-dependent NF-?B activationWe next treated A549 cells with increasing doses of CP to generate the cells bearing different extents of resistance.In the viable cells that sustain the escalating dosage of CP,cytoplasmic but not nuclear RAP1 expression was gradually induced,supporting our hypothesis that cytoplasmic RAP1 marks CP resistance.Moreover,NF-?B activity was also gradually induced,demonstrated by increasing pp65 and p-I?B? and transcription of downstream genes.Notably,the increase of pp65 and p-I?B? showed a delay compared with RAP1,suggesting their roles as the responders to RAP1 when encountering CP in the environment.Following that,we applied the sequential CP treatment to RAP1-overexpressing and RAP1-deleted cells.RAP1-overexpressing cells displayed an enhanced baseline NF-?B activity,which was further strengthened by CP.However,at the latest time point of CP treatment,there was no significant difference of NF-?B activity between the RAP1-overexpressing and the untransduced cells,indicating a saturated activity of NF-?B signaling.In contrast,as low as 0.5?M of CP showed considerable toxicity to RAP1-deleted cells,and 1?M of CP eliminated almost all the cells.Both concentrations have minimal toxicity in normal A549 cells.More importantly,in the viable RAP1-deleted cells that survived CP treatment,we failed to detect an induced NF-?B signaling.Therefore,RAP1 is confirmed as an intermediate mediator between CP treatment and NF-?B activation,which is necessary for the cells surviving CP treatment.Further,given the observation by previous studies that NF-?B directly regulates the transcription of BCL-2(30),we conclude that the RAP1-NF?BBCL2 axis mediates CP resistance in NSCLC cells.Part 6 RAP1 deletion is lethal to Cisplatin-resistant NSCLC cellsHaving demonstrated the role of RAP1 in inhibiting CP-induced apoptosis,we then switched to the other direction,investigating the possibility to target RAP1 in NSCLC cells with established CP resistance.We generated CP-resistant A549 cells(A549R)using protocols described previously(31,32).A549 R cells displayed up-regulated cytoplasmic RAP1 as well as induced NF-?B signaling.Further,the BCL-2 expression was much higher in A549 R cells compared with parental A549 cells,probably contributed by the continuous CP treatment for maintaining resistance.We then sought to attenuate CP resistance in A549 R cells through deleting RAP1.Surprisingly,lethality was observed almost immediately after applying lentiviruses encoding sh-RAP1 to A549 R cells,shown by both CCK-8 viability assay and microscopic visualization.To rule out the possibility that the CP used to maintain resistance might induce mortality,we cultured CP-res cells in CP-free media for 24 h before inducing RAP1 deletion.Such “CP clearance” did not affect the tolerance of the cells to CP,but RAP1-depleted cells still cannot survive for 7 days.Further test showed that although scramble shRNA-transduced A549 R cells had minor induction of apoptosis in response to CP treatment,A549 R cells displayed an up-regulation of cleaved caspase-3 and the almost complete loss.The expression of cleaved caspase-3 and BCL-2 in RAP1-deleted A549 R cells was similar to that in CP-treated RAP1-deleted A549 cells.The induced apoptosis was also shown by Annexin-V staining.Therefore,We found an unexpected effect of RAP1 deletion in the A549 R cells through hyper-activation of apoptosis to induce lethality,for which further CP treatment is even no longer required.Conclusions: Cisplatin(CP)is a commonly used anticancer drug that causes apoptosis by inducing DNA damage.In NSCLC cells,the resistance to CP is closely related to RAP1 in the cytoplasm.The cytoplasmic RAP1 is upregulated after CP treatment,which mediates the activation of the NF-?B signaling;the activated NF-?B regulates the downstream target gene transcription,which includes the important inhibitor of apoptosis BCL-2.RAP1 activation is likely to be part of the DNA damage response in cells.Therefore,RAP1 activates NF-?B signaling to induce the up-regulation of anti-apoptotic protein BCL-2,thereby inhibiting DNA damage induced apoptosis without significantly altering DNA damage.These findings suggest that cytoplasmic RAP1 may have important clinical significance in the treatment of cisplatin-resistant cancer.First,RAP1 can be used as an important marker for the poor prognosis of NSCLC.Second,RAP1 can be used to identify whether NSCLC is suitable for CP treatment.Finally,targeted therapy against RAP1 is a potential approach for combinatorial therapy with CP. |
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