Molecular Mechanisms And Its Function Of Tpp1 Knockdown Initiated DNA Damage Response In Tumorigenesis

Background:Telomeres are composed of specialized repeat DNA sequences bound by a core essential protein complex at the termini of all the eukaryotic chromosomes, play crucial roles in protecting chromosomes from enzymatic degradation, preventing telomere ends from being inappropriately recognized as double strand DNA breaks (DSBs), whereas initiating DNA damage response (DDR) and abnormal DNA repair, and inhibiting chromosomes end-to-end fusion, recombination and loss. Functional and structural abnormality of telomeres is tightly associated with human aging, hereditary diseases and tumors. Therefore, understanding the molecular mechanisms and investigating its role of dysfunctional telomeres becomes an active area.Telomere binding proteins play critical roles in maintaining chromosome stability and length regulation. They are composed of six essential core proteins called Shelterin, including Pot1, TRF1, TRF2, TIN2, Rap1 and TPP1. Among which, TRF1 and TRF2 are double-stranded binding proteins, Pot1 recognizes telomeric single-stranded DNA, while TPP1 recruits Pot1 to telomeres and interacts with Pot1. Furthermore, TPP1 is a TIN2-interacting protein which connects TRF1 and TRF2, contributing to the structural formation of Shelterin. Shelterin plays important roles in the specific"T"–loop structure capping telomeres ends and maintenance telomeric DNA being inappropriately recognized as double stranded breaks.One of the core proteins of the Shelterin, TPP1, was identified and characterized by three independent groups in 2004. The experimental results demonstrated that TPP1 was able to interact with Pot1, TIN2, and function as protecting the single- stranded overhang. On the other hand, TPP1 was found to physically interact with telomerase through its OB fold, and regulates telomerase enzymatic activity. However, whether TPP1 plays critical roles in preventing telomeres from being inappropriately recognized as DSBs, whereas initiates DDR and maintaining the chromosomal stability as well as suppressing cellular transformation and tumor formation in vivo, is not well understood.Objective:To study the effects on Pot1 localization at telomeres, telomere function, chromosomal stability and tumor formation in the absence of TPP1, and to investigate the molecular mechanisms and function of the signaling transduction pathways involved in lacking TPP1.Methods:1. Construct of shTPP1: A retrovirus vector encoding shRNA targeting TPP1, termed shTPP1 was constructed, and co-transfected with plasmid expressing exogenous TPP1 in 293T cells. Western blot was applied to detect the knockdown efficiency of exogenous TPP1 protein expression and RT-PCR was used to determine the endogenous TPP1 mRNA in mouse embryonic fibroblasts (MEFs) by depletion of TPP1.2. Effects on localization at telomeres of Pot1 due to TPP1 depletion: After mouse embryonic fibroblasts (MEFs) were transfected with shTPP1, plasmids encoding exogenous Pot1a or Pot1b were introduced into the MEFs with loss of TPP1. Western Blot was performed to examine the exogenous TPP1 overexpression and localization of Pot1a and Pot1b at telomeres in MEFs with knockdown of TPP1 were determined by Immunofluorescence plus Peptide Nucleic Acids- Fluorescence In Situ Hybridization (IF/PNA-FISH).3. Effects on telomere ends protection due to inhibition of TPP1: FITC conjugated Terminal deoxy-Uridine transferase (TdT-FITC) was performed to detect the chromosomal end protection in MEFs with knockdown of TPP1.4. Telomeric DNA damage response induced by TPP1 depletion: Telomere- dysfunction-induced-foci (TIF) formation was determined by IF/PNA-FISH assay in ATM+/+ and ATM-/- MEFs with loss of TPP1, respectively. Phosphorylated H2AX (γ-H2AX), one of the markers of damaged DNA was analyzed in MEFs lacking TPP1 by Western Blot.5. TPP1 depletion initiated ATM-p53 signaling transduction pathway: Cell proliferation was examined by cell growth curve and bromo- deoxyuridine (BrdU) incorporation after loss of TPP1 in primary MEFs(ATM+/+ , p53+/+). Cellular senescence was detected by the presence of the senescence-associatedβ-galactosidase (SA-β-gal) in cells. Phosphorylated p53 and induction of p21 were detected by Western Blot.6. Loss of TPP1 initiated chromosomal instability in ATM knockout and p53 knockout MEFs (ATM-/-): ATM-/- MEFs were infected with retrovirus encoding shTPP1, and then chromosomal instability and Anaphase Bridge were examined by PNA-FISH and DAPI staining.7. Cellular transformation driven by chromosomal instability and tumor formation in vivo: Soft agar assay was performed to investigate whether the elevated genomic stability in ATM-/- cells with knockdown of TPP1 (ATM-/--shTPP1) were able to initiate cellular transformation. Furthermore, the transformed ATM-/--TPP1 MEFs were injected subcutaneously into SCID mice to investigate whether transformed cells were sufficient to form tumor in vivo. PNA-FISH was performed to examine the chromosomal instability in tumor cells generated from transformed ATM-/-- shTPP1 MEFs.Results:1. Retrovirus vector encoding shRNA targeting TPP1 was successfully constructed, named shTPP1. Western Blot result showed exogenous TPP1 was significantly inhibited by shTPP1, and endogenous TPP1 mRNA of MEFs expressing shTPP1 was significantly reduced.2. IF/PNA-FISH data showed that knockdown of TPP1 prevents exogenous Pot1a and Pot1b from localizing at telomere, and TdT assay results indicated that TdT-FITC positive cells were detected in 50% of TPP1 knockdown MEFs, co-localizing with telomeres signals.3. Knockdown of TPP1 induced DNA damage marker, such asγ-H2AX and 53BP1, formed TIF at telomeres in ATM+/+ cells, but not in ATM-/- cells. Furthermore, Western Blot suggested that phosphorylated H2AX (γ-H2AX) was increased significantly in ATM+/+ cells, but not in ATM-/- cells.4. Depletion of TPP1 in primary p53+/+ MEFs resulted in cell growth arrest and increased cellular senescence associatedβ-gal staining positive cells. Western Blot demonstrated that loss of TPP1 initiated p53 phosphorylation and induction of p21 expression.5. Knockdown of TPP1 in ATM-/- cells resulted in severe chromosomal instability, anaphase bridge and multiple aberrant chromosomes, including end-to-end chromosomal fusions, such as p-p arm fusions, q-q arm fusions, p-q arm fusions and as well as diplochromosomes.6. ATM-/- cells with loss of TPP1(ATM-/--shTPP1) initiated cellular transformation and formed transformed colonies in soft agar, but not in ATM-/- cells with vector (ATM-/--vector) or ATM+/+ cells with loss of TPP1(ATM+/+-shTPP1).7. Transformed ATM-/--shTPP1 initiated tumor formation in SCID mice, and tumor cells derived from ATM-/--shTPP1 possess multiply aberrant chromosomes, consistent with those phenotypes observed in the transformed ATM-/--shTPP1 cells.Conclusions:1. Retrovirus vector encoding shRNA targeting TPP1 was successfully constructed, and shTPP1 can efficiently knock down TPP1 expression.2. TPP1 is required for Pot1a and/or Pot1b localization at telomeres, and loss of TPP1 leads to open-access telomere ends, resulting telomere dysfunction.3. Depletion of TPP1 induced ATM-dependent DDR, initiating p53-dependent cellular senescence.4. Removal of TPP1 in ATM-/- cells resulted in chromosomal instability, aberrant chromosomes with anaphase bridge formation, which is a hallmark of telomere dysfunction.5. Telomere dysfunction as a result of TPP1 depletion promotes chromosomal instability-driven cellular transformation and tumorigenesis in the absence of an ATM-dependent DDR-p53 signaling pathway.Significance:1. shTPP1 provides a useful tool to dissect TPP1 loss of function in cells.2. Loss of TPP1 results in dysfunctional telomeres and initiates ATM dependent DDR at telomeres to induce p53-mediated cellular senescence to suppress cellular transformation and tumorigenesis.3. TPP1 depletion resulting in dysfunctional telomeres, cooperating with the loss of ATM-p53-senescence signaling pathway, promotes chromosome instability, cellular transformation and tumorigenesis.4. TPP1 plays critical roles in maintaining the telomere structure and preventing telomere from being inappropriately recognized as broken DNA, which initiates DDR and abnormal DNA repair, and eventually leading to chromosome instability-driven cellular transformation and tumorigenesis.5. ATM-DDR-p53-mediated cellular senescence signaling pathway might provide a new candidate approach for tumor therapy.