RP11-81H3.2 Acts as an Oncogene via microRNA-490-3p Inhibition and Consequential Tankyrase 2 Up-Regulation in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a serious threat to human lives and is usually diagnosed at the late stages. Recently, there has been a rapid advancement in the treatment options for HCC, but novel therapeutic targets are still needed, especially for precision medicine. AIMS: We aimed to investigate the involvement of non-coding RNA RP11-81H3.2 in HCC. METHODS: The expression of RP11-81H3.2 was examined in the blood samples of HCC patients, and in the human HCC cell lines, including HepG2, Smmc-7721, and Huh7. Cell proliferation was determined using the CCK-8 and EdU assay, and cell invasion and migration were determined using the transwell/wound healing assay. The effects of RP11-81H3.2 knockdown on in vivo tumor growth were evaluated utilizing the nude mice HepG2 tumor xenograft model. RESULTS: Here, we have identified a long non-coding RNA, RP11-81H3.2, which is enriched in HCC and can promote its proliferation, migration, and invasion both in vitro and in vivo. In addition, our results showed that RP11-81H3.2 binds to and regulate miR-490-3p expression in the HCC cells. Moreover, we found that RP11-81H3.2 regulates the expression of TNKS2 via miR-490-3p. Further, we found that RP11-81H3.2 and miR-490-3p form a regulatory loop; the release of RP11-81H3.2 leads to the suppression of miR-490-3p expression, thus, further enhancing the expression of RP11-81H3.2. CONCLUSIONS: Our data have provided a novel target for the diagnosis and treatment of HCC, and sheds light on the lncRNA-miRNA regulatory nexus that can control the HCC related pathogenesis.

miR-490-3p inhibits the growth and invasiveness in triple-negative breast cancer by repressing the expression of TNKS2.

Identification of key genes driving the aggressiveness of triple-negative breast cancer (TNBC) is important to develop effective therapies. In this study, we examined the expression and biological roles of microRNA (miR)-490-3p in TNBC. Our data showed that miR-490-3p-3p was underexpressed in TNBC compared with non-TNBC tissues (P=0.0021). Similarly, this miRNA was expressed at lower levels in TNBC cell lines than in non-TNBC cell lines. Gain-of-function studies revealed that miR-490-3p-3p overexpression inhibited cell growth and invasion in both MDA-MB-231 and MDA-MB-436 TNBC cells and impaired tumorigenesis of MDA-MB-231 cells in nude mice. Mechanistically, we found that miR-490-3p negatively regulated the expression of tankyrase 2 (TNKS2) via binding to its 3'-untranslated region and then blocked the activation of ß-catenin signaling. Importantly, overexpression of a miR-490-3p-resistant form of TNKS2 reversed miR-490-3p-mediated suppression of TNBC cell proliferation and invasion. Knockdown of TNKS2 via small interfering RNA technology was found to mimic the suppressive activity of miR-490-3p in MDA-MB-231 cells. Taken together, miR-490-3p is downregulated in TNBC and plays a suppressive role in cancer cell proliferation, invasion, and tumorigenesis. The tumor suppressive activity of miR-490-3p is largely mediated through downregulation of TNKS2 and inactivation of ß-catenin signaling. Thus, miR-490-3p may represent a potential therapeutic target for TNBC.

Tankyrases regulate glucoregulatory mechanisms and somatic growth via the central melanocortin system in zebrafish larvae.

The central melanocortin system is a key regulator of energy homeostasis. Recent studies indicate that tankyrases (TNKSs), which poly(ADP-ribosyl)ate target proteins and direct them toward proteasomal degradation, affect overall metabolism, but the exact molecular mechanisms remain unclear. We used zebrafish larvae as a model to study the mechanisms by which TNKS1b, the zebrafish ortholog of mammalian TNKS1, regulates glucose homeostasis and somatic growth. In situ hybridization revealed that TNKS1b mRNA is prominently expressed in the hypothalamus and pituitary of the embryonic and larval brain. In the pituitary, TNKS1b is coexpressed with pro-opiomelanocortin a (pomca) gene in corticotropes and melanotropes. Knockdown of TNKS1b reduced the linear growth of the larvae, stimulated insulin gene and glucose transporter 4 protein, and suppressed gluconeogenic phosphoenolpyruvate carboxykinase 1 gene. This result indicates rapid glucose utilization and reduction of gluconeogenesis in TNKS1b-deficient larvae. Knockdown of TNKS1b down-regulated pomca expression and diminished α-melanocyte-stimulating hormone in the pars intermedia. Furthermore, down-regulation of TNKS1b suppressed the expression of melanocortin receptor 3 and increased the expression of melanocortin receptor 4. The collective data suggest that TNKS1b modulates glucoregulatory mechanisms and the somatic growth of zebrafish larvae via the central melanocortin system.

Expression of TNKS1 is correlated with pathologic grade and Wnt/ß-catenin pathway in human astrocytomas.

Astrocytoma is the most common neoplasm of the central nervous system, and its malignancy is closely related to activation of the Wnt/ß-catenin pathway. Accumulated evidence shows that tankyrase (TNKS) is necessary for the Wnt/ß-catenin pathway, stabilizing ß-catenin, and that TNKS1, a major member of the TNKS family, is involved in a wide range of human cancers. However, the expression of TNKS1 and the molecular relationship between TNKS1 and ß-catenin in human astrocytomas is largely unknown. In the present study, we investigated the expression of TNKS1 in human astrocytomas using reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. The mRNA and protein expression levels of TNKS1 in astrocytomas were significantly higher compared with the normal brain tissues. Significant association between TNKS1 upregulation and pathological grade of astrocytomas was also confirmed. In addition, ß-catenin immunostaining of the sampled tissues revealed a highly similar change to TNKS1. This study provides additional evidence for the involvement of TNKS1 gene and the Wnt/ß-catenin signaling pathway in the genesis and progression of astrocytoma. TNKS1 may have a key role in astrocytomas.

Human tankyrases are aberrantly expressed in colon tumors and contain multiple epitopes that induce humoral and cellular immune responses in cancer patients.

PURPOSE: Tankyrases 1 and 2 are telomere-associated poly(ADP-ribose) polymerases (PARP) that can positively regulate telomere elongation and interact with multiple cellular proteins. Recent reports implicated tankyrases as tumor antigens and potential targets of anticancer treatment. We examined expression of tankyrases in colon tumors and immune response to these enzymes in patients with different types of cancer. METHODS: mRNA and protein expression was evaluated by quantitative real-time RT-PCR and Western blotting, respectively. Humoral immune response to recombinant tankyrases was investigated by modified enzyme-linked immunoassays. Cellular immune response was analysed by ELISPOT and (51)Cr release assays. RESULTS: We found that both mRNA and protein levels of tankyrase 2 (TNKL) are upregulated in colon tumors. In contrast, protein level of tankyrase 1 (TNKS) is downregulated, while mRNA level shows variable changes. More than a quarter of colon cancer patients develop humoral immune response to at least one of the two tankyrases. In this study we mapped common and unique B-cell epitopes located in different domains of the two proteins. Additionally, we present evidence for T-cell responses both to epitopes that are unique for TNKL and to those shared between TNKL and TNKS. CONCLUSION: Our study favors a biomarker usage of antibody response to tankyrases. Spontaneous CD8(+) T-cell responses to these enzymes are rare and further investigation is needed to evaluate tankyrases as potential targets for cancer immunotherapy.

The role of the poly(ADP-ribose) polymerase tankyrase1 in telomere length control by the TRF1 component of the shelterin complex.

Tankyrase1 is a multifunctional poly(ADP-ribose) polymerase that can localize to telomeres through its interaction with the shelterin component TRF1. Tankyrase1 poly(ADP-ribosyl)ates TRF1 in vitro, and its nuclear overexpression leads to loss of TRF1 and telomere elongation, suggesting that tankyrase1 is a positive regulator of telomere length. In agreement with this proposal, we show that tankyrase1 RNA interference results in telomere shortening proportional to the level of knockdown. Furthermore, we show that a tankyrase1-resistant form of TRF1 enforced normal telomere length control, indicating that tankyrase1 is not required downstream of TRF1 in this pathway. Thus, in human cells, tankyrase1 appears to act upstream of TRF1, promoting telomere elongation through the removal of TRF1. This pathway appears absent from mouse cells. We show that murine TRF1, which lacks the canonical tankyrase1-binding site, is not a substrate for tankyrase1 poly(ADP-ribosyl)sylation in vitro. Furthermore, overexpression of tankyrase1 in mouse nuclei did not remove TRF1 from telomeres and had no detectable effect on other components of mouse shelterin. We propose that the tankyrase1-controlled telomere extension is a human-specific elaboration that allows additional control over telomere length in telomerase positive cells.

Distribution of Tankyrase-1 mRNA expression in colon cancer and its prospective correlation with progression stage.

We tested Tankyrase-1 mRNA expression in colon cancer patients to evaluate the prognostic role of this parameter by real-time RT-PCR in a retrospective group of 82 unselected patients with colon cancer. Paired cancer and corresponding not affected tissues were used. Laser-assisted microdissection was used to measure Tankyrase-1 mRNA in homogeneous cancer cell populations and in normal colon epithelium of the same patients. Tankyrase-1 mRNA in colon cancers, as a mean, was significantly higher than in paired not affected tissues (p<0.0001), but its level correlates inversely with a cancer progression stage. Survival analysis indicated that lower Tankyrase-1 mRNA expression in colon cancers was significantly associated to reduced patient survival (p=0.019) and disease-free interval (p=0.035), confirmed also in a multi-variate analysis.

hTERT, the catalytic component of telomerase, is downregulated in the haematopoietic stem cells of patients with chronic myeloid leukaemia.

Telomere shortening is associated with disease progression in chronic myeloid leukaemia (CML). To investigate the biology and regulation of telomerase in CML, we evaluated expression of the telomerase components, its regulators and several telomeric-associated proteins. Quantitative real-time-polymerase chain reaction (PCR) was used to compare gene expression in the CD34+/leukaemic blast cells of 22 CML patient samples to the CD34+ cell population of healthy individuals. hTERT, the catalytic component of telomerase, was downregulated in eight of 12 chronic phase (CP) patients (P = 0.0387). Furthermore, hTERT was significantly downregulated in two of three patients in accelerated phase (AP) and seven of seven patients in blast crisis (BC), P = 0.0017. Expression of hTR and telomeric-associated proteins TEP1, TRF1, TRF2, tankyrase and PinX1 was high in the majority of CP and AP patients. With the exceptions of TEP1 and hTR, expression of these factors was highest in CP and decreased during disease progression. Expression of c-Myc, a positive regulator of hTERT transcription, correlated with hTERT expression and decreased with disease progression, falling below control levels in BC. hTERT levels were increased in CP patients following successful treatment with imatinib, relative to untreated CP patients. We suggest that reduced hTERT expression directly causes the shortened telomeres observed in CML.

The expression of telomeric proteins and their probable regulation of telomerase during the differentiation of all-trans-retinoic acid-responsive and -resistant acute promyelocytic leukemia cells.

Telomerase activity has been linked to retinoid induction of tumor cell differentiation, and the patterns of telomerase expression are different in the 2 pathways of acute promyelocytic leukemia (APL) cell differentiation: the retinoic acid receptor 3 (RAR3)-dependent and the retinoic X receptor 3 (RXR3)-dependent pathways. Still, whether telomeric proteins respond to retinoid treatment is not clear. If they do, how they would respond and how they would interfere in telomerase regulation during differentiation are also unclear. Using all-trans-retinoic acid (ATRA)-sensitive and -resistant APL cell lines NB4, NB4-R1, and NB4-R2, we analyzed a panel of telomeric proteins, including TRF1, PINX1, TANK1, and TANK2, at the messenger RNA (mRNA) and protein expression levels during the differentiation of these cell lines in the 2 pathways. Our analyses showed that both mRNA and protein expression of TRF1 remained stable during NB4 and NB4-R1 cell differentiation but slightly increased in NB4-R2 cells, suggesting that TRF1 may have different functions in the RAR3- and RXR3-dependent pathways. The stable expression of TRF1 may be because telomere length remains unchanged. Pinx1 mRNA expression was tightly correlated with telomerase reverse transcriptase (hTERT) mRNA expression during differentiation. Variation in Pinx1 expression may be a reaction induced by hTERT expression variation. TANK1 mRNA expression and TANK1 protein levels were both down-regulated in all 3 APL cell lines at a later period of differentiation, suggesting that TANK1 may positively regulate telomerase activity and that both RAR3- and RXR3-dependent pathways may exert this regulation.TANK2 expression levels remained stable in all 3 APL cell lines during differentiation, showing that TANK2 may have little effect on telomerase. Thus, our studies provide an outline of the dynamics of telomeric protein expression and the probable regulatory effects of these proteins on telomerase during the differentiation of ATRA-responsive and -resistant APL cells.

Tankyrase, a positive regulator of telomere elongation, is over expressed in human breast cancer.

Tankyrase promotes telomere elongation by interaction with the telomeric protein binding factor TRF1, a negative regulator of telomere extension. We measured tankyrase mRNA by real-time RT-PCR in 66 breast cancers and in paired normal tissues. Results were compared with hTERT mRNA expression. The levels of tankyrase in breast cancers were significantly higher in comparison to normal tissues (P<0.0001) and significantly related to the status of progesterone receptors. No relationship was found between tankyrase and hTERT mRNA expression in breast cancers. According to our results, tankyrase expression appeared up regulated in breast cancers.

Antisense inhibition of Bcr-Abl/c-Abl synthesis promotes telomerase activity and upregulates tankyrase in human leukemia cells.

Clinical studies in chronic myelogenous leukemia demonstrate that the overexpression of Bcr-Abl tyrosine kinase is usually accompanied by relatively low telomerase activity in the chronic phase, which reverts to a high activity in blast crisis. The present study was designed to investigate the cross-talk between both enzymes, using Bcr-Abl-positive K-562 and Bcr-Abl-negative Jurkat cell lines, treated with antisense oligodeoxyribonucleotides (ODNs) against Bcr-Abl/c-Abl mRNA. The decreased amount and enzyme activity of Bcr-Abl/c-Abl provoked telomerase activation in both cell lines. After short-term treatment with anti-Bcr-Abl/c-Abl ODNs (6 days), no variations in hTERT and phospho-hTERT were detected. The decreased amount of Bcr-Abl/c-Abl was accompanied by: alterations in telomeric associated proteins-overexpression of tankyrase and decreased amount of TRF1/Tin2, cell growth arrest of K-562 cells, reaching a plateau after 6 days treatment, and increased proliferating activity of Jurkat cells. No changes in telomere length were detected after short-term treatment. In contrast, after long-term treatment with anti-Bcr-Abl/c-Abl ODNs (36 days), a significant elongation of telomeres and enhancement of hTERT were established, accompanied by an increased proliferating activity of both cell lines. These data provide evidence that the inhibition of Bcr-Abl or c-Abl synthesis keeps a potential to restore or induce cell proliferation through telomere lengthening control and telomerase activation.

Protection of internal (TTAGGG)n repeats in Chinese hamster cells by telomeric protein TRF1.

Chinese hamster cells have large interstitial (TTAGGG) bands (ITs) which are unstable and should be protected by an unknown mechanism. Here, we expressed in Chinese hamster V79 cells green fluorescent protein (GFP)-tagged human TRF1, and found that a major fraction of GFP-TRF1 bound to ITs is diffusionally mobile. This fraction strongly decreases after treatment of cells with wortmannin, a protein kinase inhibitor, and this drug also increases the frequency of chromosome aberrations. Ionizing radiation does not induce detectable translocation of GFP-TRF1 to the sites of random double-strand breaks visualized using antibodies against histone gamma-H2AX. TRF1 is known to be eliminated from telomeres by overexpression of tankyrase 1 which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding tankyrase 1 and found that the frequency of chromosome rearrangements is increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 is involved in sequence-specific protection of internal nontelomeric (TTAGGG)n repeats.

[Recognition of internal (TTAGGG)n repeats by telomeric protein TRF1 and its role in maintenance of chromosomal stability in Chinese hamster cells]. / Uznavanie vnutrennikh povtorov (TTAGGG)n telomernym belkom TRF1 i ego rol' v podderzhanii stabil'nosti khromosom v kletkakh kitaiskogo khomiachka.

Mammalian telomeres contain long tandem (TTAGGG)n repeats, which are protected by a complex of different proteins. Telomeric repeat-binding factors TRF1 and TRF2 play the key role in protection of telomeres through the formation of terminal loops (called T-loop). A T-loop isolates the 3' strand telomeric end and with this mechanism protects telomeres from the influence of enzymes of DNA reparation and telomere fusions and also interferes with the interaction of telomerase with telomeres. Many vertebrate species also contain large blocks of (TTAGGG)n sequences in pericentric and interstitial chromosome bands. The Chinese hamster genome contains a total of 18 arrays of these non-telomeric internal (TTAGGG)n sequences (ITs). Chromosome bands containing these arrays are unstable and should be protected with the help of another mechanism, rather than that using telomeres. In this study we analysed association of Green Fluorescent Protein (GFP)-tagged TRF1 in Chinese hamster V79 cells with ITs. We found that in these cells GFP-TRF1 associates with ITs in the interphase nucleus. We detected a little overlap between IT-associated GFP-TRF1 and random DSB sites visualized after the treatment of V79 cells with ionizing radiation. We found that the treatment of V79 cells with WM significantly increases the frequency of spontaneous chromosome aberrations. These WM effects are possible due to inhibiting phosphorylation of TRF1 by ATM. TRF1 is known to be eliminated from telomeres by overexpression of TANK1, which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding TANK1 and found that the frequency of chromosome rearrangements increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 may be involved in the sequence-specific protection of internal non-telomeric (TTAGGG)n repeats.

Down-regulation of TRF1, TRF2 and TIN2 genes is important to maintain telomeric DNA for gastric cancers.

BACKGROUND: The maintenance of telomeres may be required for long-term proliferation of tumors. Activity of telomerase, a ribonucleoprotein complex that elongates telomeres, has been found in almost all human tumors but not in adjacent normal cells. Several factors which regulate telomere length, TRF1 and 2, TIN2, tankyrase and Rap1, have been identified. TRF1, TRF2 and TIN2 are negative regulators of telomere length, while tankyrase and Rap1 act as positive regulators. In this study, we quantitated the mRNA of these five genes in gastric cancers to clarify the mechanism by which cancer cells maintain telomere length. MATERIALS AND METHODS: The expression of these five genes transcription was determined using a quantitative RT-PCR. RESULTS: TRF1, TRF2 and TIN2 mRNAs were significantly down-regulated in cancers compared to non-cancerous mucosa. Neither tankyrase nor Rap1 was upregulated in cancers. CONCLUSION: Down-regulation of TRF1, TRF2 and TIN2 gene expression may be important to maintain telomeres in gastric cancer.