The Anti-cancer Effects Of Disrupting The TPP1-mediated Recruitment Of Telomerase To Telomere In NSCLC

Research objective:The stabilization of the telomere is essential for the development of tumors.Human telomeres are composed of the repeat DNA sequence(TTAGGG)and the protein-complex called shelterin,which plays a key role in maintaining and protecting telomeres.Shelterin comprises TRF1,TRF2,POT1,TIN2,TPP1 and RAP1.TPP1 protects telomeres by linking the telomere double-stranded region with the telomere single-stranded region by binding to TIN2 and POT1.In addition,TPP1 binds to telomerase and recruits it to the chromosomal end to synthesize telomeric DNA.Any mutantion of the telomerase-binding domain of TPP1 lost the function of prolonging telomeres and the proliferation of cells in tumors is inhibited so that the inhibition of telomerase-recruitment will represent a new anti-tumor strategy targeting telomeres.Although researchers have elucidated the biological functions of TPP1 involved in the regulation of telomere during the past decades,the significance of the methods to block the TPP1-mediated pathway about telomerase recruitment in anti-tumor therapy is not well defined.We intend to block the TPP1-mediated pathway that the telomerase is recruited to the telomere by overexpessing the polypeptide of telomerase-binding domain of TPP1 to competitively inhibit the binding between TPP1 and telomerase and explore whether the strategy could be utilized in the treatment of non-small cell lung cancer potentially.Research method:As an important member of the shelterin complex,TPP1 can bind to the telomerase catalytic subunit,TERT,with its OB domain(TPP1 telomerase binding domain)to recruit telomerase to the telomere end to synthesize telomere DNA.In this study,we used the methods that overexpressing the TPP1-OB domain to competitively inhibit the endogenous TPP1-TERT interaction,and then block the recruitment of telomerase to the end of the chromosome for telomere synthesis without interfering with the telomere protection function of TPP1,to investigate the inhibition of the targeting strategy on the proliferation of lung cancer cells in vitro and in vivo.Firstly,it was confirmed by immunoprecipitation experiment that the exogenous overexpressing of TPP1-OB could inhibit the binding of endogenous TPP1 with TERT.Then,the lung cancer cell lines which could stably overexpress TPP1-OB were constructed.Short-term cell proliferation assay,long-term cell proliferation assay,drug sensitivity assay and plate-cloning assay were used to explore the effects on the cell proliferation and clonality.The sensitivity of chemotherapeutic drugs,cell aging and apoptosis detection and cell cycle analysis were used to study the effects on cell senescence,apoptosis or cell cycle.Further,soft agar colony formation experiment and nude-mice xenograft models were to examine the effect of TPP1-OB on cell colony forming ability and tumor growth in vivo.Research result:Overexpression of TPP1-OB could competitively inhibit the binding of endogenous TPP1 to TERT,which prevented telomerase from reaching the telomere end,thereby blocking the telomerase-telomere recruitment pathway in cells.In cell lines of non-small cell lung cancer,overexpression of TPP1-OB resulted in decreasing long-term proliferative capacity and platelet colony formation by shortening telomere length.TPP1-OB inhibited cell proliferation mainly through the blockade of G1 in cell cycle and the increasing apoptosis.Soft agar colony formation experiments showed that TPP1-OB could inhibit the colony forming ability in lung cancer cells.And,TPP1-OB could also inhibit the growth of lung cancer cells in vivo.Besides,we also found that TPP1-OB could enhance the sensitivity of lung cancer cells to the chemotherapy drug paclitaxel.Conclusion:Our study demonstrated that the overexpression of TPP1-OB protein could inhibit the telomerase-recruitment pathway mediated by TPP1,which could become a potential new strategy to target the telomere applied into the anti-lung-cancer therapy.