Wenrui Duan

Methylation of Adjacent CpG Sites Affects Sp1/Sp3 Binding and Activity in the p21Cip1 Promoter

ABSTRACT DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C→W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. By analyzing luciferase activity of transfected p21Cip1 promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21Cip1 expression. We hypothesize that hypermethylation may interfere with Sp1/Sp3 binding. By using an electrophoretic mobility shift assay, we show that, although methylation within the consensus Sp1-binding site did not reduce Sp1/Sp3 binding, methylation outside of the consensus Sp1 element induced a significant decrease in Sp1/Sp3 binding. Depsipeptide induced p21Cip1 expression was reconstituted when cells were pretreated with 5-aza-2′-deoxycytidine. Our data suggest, for the first time, that hypermethylation around the consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, therefore leading to a reduced p21Cip1 expression in response to depsipeptide treatment.

Increased expression of unmethylated CDKN2D by 5-aza-2′-deoxycytidine in human lung cancer cells

DNA hypermethylation of CpG islands in the promoter region of genes is associated with transcriptional silencing. Treatment with hypo-methylating agents can lead to expression of these silenced genes. However, whether inhibition of DNA methylation influences the expression of unmethylated genes has not been extensively studied. We analysed the methylation status of CDKN2A and CDKN2D in human lung cancer cell lines and demonstrated that the CDKN2A CpG island is methylated, whereas CDKN2D is unmethylated. Treatment of cells with 5-aza-2′-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methyltransferase 1, induced a dose and duration dependent increased expression of both p16INK4a and p19INK4d, the products of CDKN2A and CDKN2D, respectively. These data indicate that global DNA demethylation not only influences the expression of methylated genes but also of unmethylated genes. Histone acetylation is linked to methylation induced transcriptional silencing. Depsipeptide, an inhibitor of histone deacetylase, acts synergistically with 5-Aza-CdR in inducing expression of p16INK4a and p19INK4d. However, when cells were treated with higher concentrations of 5-Aza-CdR and depsipeptide, p16INK4a expression was decreased together with significant suppression of cell growth. Interestingly, p19INK4d expression was enhanced even more by the higher concentrations of 5-Aza-CdR and depsipeptide. Our data suggest that p19INK4d plays a distinct role from other INK4 family members in response to the cytotoxicity induced by inhibition of DNA methylation and histone deacetylation.

Precancerous Stem Cells Have the Potential for both Benign and Malignant Differentiation

Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors—namely, precancerous stem cells (pCSCs) —have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.

P21 response to DNA damage induced by genistein and etoposide in human lung cancer cells.

The p21(WAF1/Cip1) gene plays a central role in cell cycle regulation. Here we show that topoisomerase II inhibitors, genistein and etoposide, induce p21(WAF1/Cip1) expression mainly in a p53-dependent manner in human lung cancer cell line A549. However, although p53 accumulated, p21(WAF1/Cip1) expression did not depend on the level of Ser15 phosphorylation of p53. Caffeine, an ataxia telangiectasia-mutated (ATM), and ATM- and Rad3-related kinase (ATR) inhibitor, abrogated genistein-induced p21(WAF1/Cip1) and largely blocked etoposide-induced p21(WAF1/Cip1) expression. Wortmannin, an ATM- and DNA-dependent protein kinase inhibitor, partially inhibited p21(WAF1/Cip1) expression induced by genistein and etoposide, whereas UCN-01, a Chk1 inhibitor, partially blocked etoposide, but not genistein-induced p21(WAF1/Cip1) expression. These data suggest that both genistein and etoposide induce p21(WAF1/Cip1) expression in a p53-dependent manner. Genistein appears to stimulate p21(WAF1/Cip1) expression through p53 via ATM, whereas etoposide may activate both ATM and ATR pathways. Our results suggest different mechanisms participate in genistein and etoposide induced p21(WAF1/Cip1) expression.

Germline stem cell gene PIWIL2 mediates DNA repair through relaxation of chromatin.

DNA damage response (DDR) is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV) irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili-/- MEFs) were defective in cyclobutane pyrimidine dimers (CPD) repair after UV treatment. As a result, the UV-treated mili-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose) polymerase (PARP) and Bik. The impaired DNA repair in the mili-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine–guanine (Pt-[GG]) and double strand break (DSB) repair were also defective in the mili-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR), respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.

MicroRNA-34a is an important component of PRIMA-1-induced apoptotic network in human lung cancer cells

Restoration of p53 function in tumor cells would be an attractive strategy for lung cancer therapy because p53 mutations are found in more than 50% of lung cancers. The small molecule PRIMA‐1 has been shown to restore the tumor suppression function of p53 and to induce apoptosis in human tumor cells. The mechanism of apoptosis induced by PRIMA‐1 remains unclear. We investigated the effects of PRIMA‐1 in apoptosis with Western immunoblot analysis, TaqMan microRNA real‐time PCR, cell viability analysis and flow cytometry using human lung cancer cell lines containing mutant (H211 and H1155), wild‐type (A549) or null (H1299) p53. PRIMA‐1 induced massive apoptosis in the H211 and H1155 cells, but was less toxic to the A549 and H1299 cells. Western immunoblot analysis showed cleavage of PARP in H211 and H1155 cells but not in A549 and H1299 cells following treatment with PRIMA‐1. In addition, p53 protein was also phosphorylated in H211 and H1155 cells. TaqMan microRNA assay showed that the expression of microRNA‐34a was increased in the H211 and H1155 cells posttreatment. Knockdown microRNA‐34a decreased the rate of apoptosis caused by PRIMA‐1. The above results suggest that microRNA‐34a is one of the important components of PRIMA‐1‐induced apoptotic network in the cancer cells harboring mutant p53.

Lung-specific expression of human mutant p53-273H is associated with a high frequency of lung adenocarcinoma in transgenic mice.

To investigate the tumorigenic potential of mutant p53 when selectively expressed in lung tissue, a transgenic mouse model was developed in which a mutant form of p53 (p53-273H) was placed under the transcriptional control of the lung-specific human surfactant protein C (SP-C) promoter. Two founder mice were identified, and a line of SP-C/p53-273H transgenic mice was established from one of the founders. Human p53-273H protein was detected specifically in lung tissue from transgenic mice. Malignant tumors, which were histologically characterized as adenocarcinomas, were observed in transgenic mice, with the earliest onset documented at 4 months of age. To further evaluate incidence and onset of tumor formation, transgenic mice (n=113) were sacrificed at age intervals ranging from 4–15 months. At 13–15 months of age, transgenic mice were significantly more likely to have lung tumors at necropsy than age-matched non-transgenic littermates (9 out of 39 (23%) versus 2 out of 35 (5.7%), χ2 test, P=0.036). The SP-C/p53-273H transgenic mice described here thus represent a genetically defined model with which to study the role of p53 mutations in lung tumorigenesis, as well as the potential complementary contributions of other genetic alterations or environmental carcinogens to lung tumor development.

Cellular localization of protein arginine methyltransferase-5 correlates with grade of lung tumors

BackgroundProtein arginine methyltransferase-5 (PRMT5) is a chromatin-modifying enzyme capable of methylating histone and non-histone proteins, and is involved in a wide range of cellular processes that range from transcriptional regulation to organelle biosynthesis. As such, its overexpression has been linked to tumor suppressor gene silencing, enhanced tumor cell growth and survival.Material and methodsQuantitative real-time polymerase chain reaction, Western immunoblot and immunohistochemistry were used to characterize PRMT5 expression in lung cancer cell lines and human tumors. Clinicopathological findings of tissue microarray based samples from 229 patients with non-small cell lung carcinomas (NSCLC) and 133 cases with pulmonary neuroendocrine tumors (NET) were analyzed with regard to nuclear and cytoplasmic PRMT5 expression.ResultsThere was statistically significant difference in PRMT5 messenger RNA expression between tumors and nonneoplastic lung tissues. Immunoblot experiments showed abundant expression of PRMT5 and its symmetric methylation mark H4R3 in lung carcinoma but not in non-neoplastic human pulmonary alveolar and bronchial epithelial cell lines. More than two thirds of lung tumors expressed PRMT5. High levels of cytoplasmic PRMT5 were detected in 20.5% of NSCLC and in 16.5% of NET; high levels of nuclear PRMT5 were detected in 38.0% of NSCLC and 24.0% of NET. Cytoplasmic PRMT5 was associated with high grade in both NSCLC and pulmonary NET while nuclear PRMT5 was more frequent in carcinoid tumors (p < 0.05).ConclusionThe observed findings support the role of PRMT5 in lung tumorigenesis and reflect its functional dichotomy in cellular compartments.Virtual slideThe virtual slides for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1611895162102528

Fanconi anemia repair pathway dysfunction, a potential therapeutic target in lung cancer.

The Fanconi anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple-staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin-embedded (FFPE) tumor samples. DNA-repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA-damaging drugs. We screened 139 non-small cell lung cancer (NSCLC) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel-targeted agents in the background of FA deficiency, we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down NSCLC cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5 μM) and BMN673 (0.5 μM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5 μM. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL-2/XL inhibitor ABT-263 at a dose of 2 μM. The treated cells were harvested at 24, 48, and 72 h post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 h post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathway plays essential roles in response to DNA damage, our results suggest that a subset of lung cancer patients are likely to be more susceptible to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. These subjects can be identified through FATSI analysis. Clinical trials to evaluate this therapeutic concept are needed.

Sensitivity to the non-COX inhibiting celecoxib derivative, OSU03012, is p21WAF1/CIP1 dependent

OSU03012 is a non‐COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21WAF1/CIP1 were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21WAF1/CIP1 were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53‐p21WAF1/CIP1 checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21WAF1/CIP1−/− mouse embryo fibroblasts were more sensitive to OSU03012‐induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21WAF1/CIP1 is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.