Pinx1 Biological Functions In The Telomere / Telomerase Regulation And Tumor Research

Telomerase maintains telomere lengths and thus has been implicated in the two important human health problems,ageing and cancers.Telomerase is a ribonucleoprotein complex with specialized reverse transcriptase activity,and is minimally composed of a single-stranded telomerase template RNA(hTR)and a reverse transcriptase protein(hTERT).hTERT serves as the telomerase catalytic subunit by reverse transcribing a template region of hTR RNA as telomeric DNA repeats which are added onto chromosomal ends thereby replenishes the loss of telomeric DNA sequence during cell division,hTERT is the rate-limiting component for human cellular telomerase regulations.Its gene expression is suppressed by multiple tumor suppressing pathways in normal human somatic cells.Consequently, normal somatic cells do not possess detectable telomerase activity and their telomeres are subjected to a progressive shortening associated with continual cell divisions and eventually evoking cellular senescence when shortening to a critical point.Cancerous mutations leading to hyperactivity of oncogenic transcriptional factors or/and loss-of-function of tumor suppressors may activate the transcription of hTERT gene, result in telomerase reactivation and thereby endowing cancer cells telomere-length maintenance and cellular immortalization.In addition to the transcriptional control, post-translational mechanisms also play crucial roles in hTERT functional regulations. It has been recently demonstrated that a mechanism on regulating the behavior of hTERT nucleolar localization in normal cells is widely disrupted in various cancer cells,suggesting that the abberant mechanism in telomerase post-translational regulations are also involved in human cancers.Moreover,how the expressed telomerase is targeted onto telomeres for function is still an unknown question in the field of the current telomere biology.Given that telomerase has been established as a valuable target for the anti-cancer therapy,further understanding the mechanisms of telomerase regulations is therefore of importance for the medical usages of this enzyme. PinX1 is an evolutionally conserved nucleolar protein that has been recently demonstrated as a telomerase/telomere interacting factor in both the humans and the yeasts.The human PinX1(PinX1)gene locates to the chromosome 8p23,a region with high frequency of loss-of-heterozygous in multiple human cancers.The PinX1 protein possessess 328 amino acids,with two independent hTERT-bindings identified to either its extream N-(aa1-142)or the C-(aa254-328)terminal polypeptide sequences.Ectopic overexpression of a PinX1 C-terminal polypeptide has been experimantally shown to be able to completely destroy telomerase activity,shorten telomere-length and inhibit both in vitro and in vivo growth in a telomerase-positive human cancer cell line,leading to the suggestion of PinX1 an intrinsic telomerase inhibitor and a putative tumor suppressor in human cells.However,the mechanisms of PinX1 on telomerase/telomere regulations and its relationship with cancers have not been intensively investigated yet.Additionally,PinX1 also directly interacts with the human telomeric protein TRF1 and locates to telomeres besides the nucleolus in cells.But the significances of the PinX1 telomere association again have not been elucided.Notably,genetic ablation of PinX1 expression has been shown to shorten, rather than to elongate,telomeres and inhibit proliferation in affected yeast cells, raising a posibility that PinX1 may perform multiple roles on telomerase/telomere regulation in cells.Given the importance of telomerase/telomere regulations in human cancers,it is necessary to further characterize the roles of PinX1 in these processes.In this present study,we investigated the roles of PinX1 in telomerase/telomere regulations as well as in cancers by focusing on the following issues:1.The roles and biological significances of PinX1 on regulation telomerase nucleolar localization.Telomerase has been shown to be subjected to a dynamic nucleolar transportation that is depended on the cell-cycle,cellular transformation and DNA damaging in human cells.But the factors involved in regulating telomerase nucleolar localization have not been identified yet.PinX1 is itself a nucleolar protein. Its interaction with hTERT indicates a possibility that it might have an effect on regulation telomerase nucleolar localization.By ectopic PinX1 overexpression,we found that it has an effect to promote both the endogenous and the ectopic expressed hTERT renucleolar accumulation in cancer cells.Furthermore,we have identified that the domain for mediating hTERT nucleolar accumulation is within the central portion of the PinX1 polypeptide suequence(aa249-254)and is unrelated to its two-known N-terminal or C-terminal hTERT-binding domain.Moreover,we demonstrate that a special PinX1 mutant(Ser254Cys,Cys265Tyr),which is present at a high frequency in human hepatoma,is defect in mediating hTERT nucleolar localization but still retains the effect of telomerase catalytic regulation.These results thus reveal a novel role of PinX1 on mediating telomerase nucleolar localization that is unrelated to the regulation of telomerase catalytic activity,and also suggest that mutational inhibition of this putative PinX1-dependent telomerase nucleolar localization may account for the abberant telomerase nucleolar exclusive localization in human cancer cells.2.PinX1 expression in human cells and its biological significances in cancer cells.The fact that PinX1 has multiple functions on telomerase regulations prompts us to further investigate its physiological roles in cancer cells.By developing specific antibodies against PinX1,we examined the expression of PinX1 protein in different cultural human cell lines and found that PinX1 is constitutively expressed in all tested human cell lines regardless of their telomerase characteristics and malignant status. Moreover,no sign of downregulation of PinX1 expression level was found in human cancerous cell lines as comared to that in normal cells,indicating that suppression of PinX1 expression is not a frequent event in human cellular carcinogenesis and could not account for the telomerase reactivation that is widely happened in almost all human cancer cells.By stable transfection of a potent shRNA specifically against PinX1,we examined the effects of PinX1 depletion on cancer cells and found that PinX1 suppression resulted in a significant telomere-length shortening in both the telomerase-positive HepG2 and HT1080 human cancer cell lines accompanied with a significant reduction of the tumorigenicity as well as the resistance to the DNA-damage induced apoptosis in these cancer cells.However,the effects of PinX1 silencing were not exhibited in the telomerase-negative U2OS cancer celt line.These results indicate that the constitutive expression of PinX1 attributes to telomere maintenance and tumorigenicity in telomerase-positive cancer cells,therefore revealing a novel positive role of PinX1 on regulating the telomerase-mediated telomere-length maintenance in human cells.3.The mechanism underlying PinX1 on facilating the telomerase-mediated telomere maintenance.How telomerase is targeted onto telomeres for action is an unknown question in the telomere biology.In above studies,we have demonstrated that PinX1 has a positive role on regulating the telomerase-mediated telomere lengthening.Since PinX1 has been shown to interact with both telomerase and telomeres,one possibility is that it might function as a linker between telomerase and telomeres in cells.TRF1 is the only known telomeric protein component for PinX1 binding.However,given TRF1 has been established as a dominant negative factor on regulating the access of telomerase to telomeres for action,it could be exclused that the putative PinX1-TRF1 interaction may serve as an avenue for delivering telomerase onto telomere for action,suggesting that other telomeric protein component might be involved in PinX1 binding for telomerase telomere targeting.By the assays of co-immunoprecipitation,we found that PinX1 can form complexes with the telomeric single-stranded DNA binding protein Pot1 in vivo.Inhibiting PinX1 expression by shRNA can significantly decrease the Pot1 associated telomerase.By stable overexpression of PinX1-Pot1 fusion proteins,which simulate a situation of PinX1-Pot1 constitutive interaction in vivo,we found that these fusion proteins were able to significantly overelongate telomere-length in telomerase-positive,but not telomerase-negative human cells.By the same time,other members of our laboratory have demonstrated a direct interaction between PinX1 and Pot1 and shown that specifically interrupting the PinX1-Pot1 interaction could remarkably shorten telomere-length in telomerase-positive cells.Taken together,our results demonstrate that the PinX1-Pot1 interaction functions as a pathway for delivering telomerase onto telomeres for action and that may account for the mechanism of PinX1 on facilitating the telomerase-mediated telomere-length maintenance in cancer cells.Conclusions:In this present study,we reveal that PinX1,the currently proposed telomerase inhibitor and a putative human tumor suppressor,actually possesses multiple roles on telomerase regulations by mediating its nucleolar localization and facilitating its access onto telomeres for action.Our data suggest that the constitutive expression of PinX1 attributes to telomere maintenance and tumorigenicity in these cancer cells under their physiological growth condition.We further identify a novel protein-protein interaction between PinX1 and the telomeric single-stranded DNA binding protein Potl and demonstrate that the PinX1-Pot1 interaction functions as a pathway for telomerase telomeric delivery,thereby underlying the mechanism of PinX1 on positively regulating the telomerase-mediated telomere-length maintenance. As silencing PinX1 as well as disruption of the PinX1-Pot1 interaction both result in telomere dysfuction in telomerase-positive cancer cells,our data also suggest that targeting the positive-role of PinX1 on telomerase/telomere regulation might represent a novel strategy for the currently ensued telomerase-based anticancer therapies.