Identification of a novel PARP4 gene promoter CpG locus associated with cisplatin chemoresistance.

The protein family of poly (ADP-ribose) polymerases (PARPs) is comprised of multifunctional nuclear enzymes. Several PARP inhibitors have been developed as new anticancer drugs to combat resistance to chemotherapy. Herein, we characterized PARP4 mRNA expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. PARP4 mRNA expression was significantly upregulated in cisplatin-resistant ovarian cancer cell lines, and this upregulation was associated with the hypomethylation of specific cytosine-phosphate-guanine (CpG) sites (cg18582260 and cg17117459) on its promoter. Reduced PARP4 expression was restored by treating cisplatin-sensitive cell lines with a demethylation agent, implicating the epigenetic regulation of PARP4 expression by promoter methylation. Depletion of PARP4 expression in cisplatin-resistant cell lines reduced cisplatin chemoresistance and promoted cisplatin-induced DNA fragmentation. The differential mRNA expression and DNA methylation status at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) according to cisplatin responses, was further validated in primary ovarian tumor tissues. The results showed significantly increased PARP4 mRNA expressions and decreased DNA methylation levels at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) in cisplatin-resistant patients. Additionally, the DNA methylation status at cg18582260 CpG sites in ovarian tumor tissues showed fairly clear discrimination between cisplatin-resistant patients and cisplatin-sensitive patients, with high accuracy (area under the curve = 0.86, P = 0.003845). Our findings suggest that the DNA methylation status of PARP4 at the specific promoter site (cg18582260) may be a useful diagnostic biomarker for predicting the response to cisplatin in ovarian cancer patients. [BMB Reports 2023; 56(6): 347-352].

SPSB1 enhances ovarian cancer cell survival by destabilizing p21.

SPRY domain-containing SOCS box protein 1 (SPSB1) is an E3 ligase adaptor protein with unknown functions in cancer cells. In this study, we found that SPSB1 knockdown markedly decreased the viability and migration of ovarian cancer cells, while ectopic SPSB1 overexpression in IL-3-dependent Ba/F3 cells significantly increased their proliferation rate compared with empty vector-transfected cells. SPSB1 knockdown significantly elevated p21 protein and mRNA levels and induced apoptosis in ovarian cancer cells, as evidenced by increased levels of cleaved PARP and decreased levels of Bcl-2. Notably, mechanistic investigations revealed that SPSB1 accelerated p21 destabilization by directly interacting with p21 and promoting its ubiquitin-mediated proteasomal degradation. Taken together, our findings provide novel insights into the role of SPSB1 in ovarian cancer cells.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer.

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Aberrant epigenetic regulation of GABRP associates with aggressive phenotype of ovarian cancer.

Metastasis is a major cause of therapeutic failure in ovarian cancer. To elucidate molecular mechanisms of ovarian cancer metastasis, we previously established a metastatic xenograft mouse model using human ovarian carcinoma SK-OV-3 cells. Using gene expression profiling, we found that γ-aminobutyric acid (GABA)A receptor π subunit (GABRP) expression was upregulated (>4-fold) in metastatic tissues from our xenograft mice compared with SK-OV-3 cells. Importantly, GABRP knockdown diminished the migration and invasion of SK-OV-3 cells, and reduced extracellular signal-regulated kinase (ERK) activation while overexpression of GABRP exhibited significantly increased cell migration, invasion and ERK activation. Moreover, treatment with the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 similarly suppressed the migration and invasion of SK-OV-3 cells, implying that GABRP promotes these cellular behaviors by activating the MAPK/ERK pathway. Using genome-wide DNA methylation profiling, we identified hypomethylated CpG sites in the GABRP promoter in metastatic tissues from the xenograft mice compared with SK-OV-3 cells. Treatment with a DNA methyltransferase inhibitor demonstrated that methylation at -963 bp from the GABRP transcription start site (-963 CpG site) was critical for the epigenetic regulation of GABRP. Finally, we analyzed human ovarian cancer patient samples and showed DNA hypomethylation at the GABRP -963 CpG site in advanced stage, but not early-stage, primary tumors compared with their paired normal tissues. These findings suggest that GABRP enhances the aggressive phenotype of ovarian cancer cells, and that the DNA methylation status of the GABRP -963 CpG site may be useful for predicting the metastatic potential in ovarian cancer patients.

Aberrant Hypomethylation of Solute Carrier Family 6 Member 12 Promoter Induces Metastasis of Ovarian Cancer.

PURPOSE: Ovarian cancer (OC) is the most fatal of gynecological malignancies with a high rate of recurrence. We aimed to evaluate the expression of solute carrier family 6, member 12 (SLC6A12) and methylation of its promoter CpG sites in a xenograft mouse model of metastatic OC, and to investigate the regulatory mechanisms that promote aggressive properties during OC progression. MATERIALS AND METHODS: Expression of SLC6A12 mRNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and DNA methylation status of its promoter CpGs was detected by quantitative methylation-specific PCR. The metastatic potential of SLC6A12 was evaluated by in vitro migration/invasion transwell assays. Gene expression and DNA methylation of SLC6A12 and clinical outcomes were further investigated from publicly available databases from curatedOvarianData and The Cancer Genome Atlas. RESULTS: SLC6A12 expression was 8.1-14.0-fold upregulated and its DNA methylation of promoter CpG sites was 41-62% decreased in tumor metastases. After treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor, the expression of SLC6A12 was profoundly enhanced (~8.0-fold), strongly supporting DNA methylation-dependent epigenetic regulation of SLC6A12. Overexpression of SLC6A12 led to increased migration and invasion of ovarian carcinoma cells in vitro, approximately 2.0-fold and 3.3-fold, respectively. The meta-analysis showed that high expression of SLC6A12 was significantly associated with poor overall survival [hazard ratio (HR)=1.07, p value=0.016] and that low DNA methylation levels of SLC6A12 at specific promoter CpG site negatively affected patient survival. CONCLUSION: Our findings provide novel evidence for the biological and clinical significance of SLC6A12 as a metastasis-promoting gene.

Synaptotagmin-like protein 2 gene promotes the metastatic potential in ovarian cancer.

Ovarian cancer (OC) metastasis has unique biological behavior and most commonly occurs via the transcoelomic route. Previously, we established a mouse xenograft model of human ovarian carcinoma and analyzed alterations in gene expression during metastasis. Among the genes that were differentially expressed more than 2-fold in the xenografts compared with the SK-OV-3 cells, we selected synaptotagmin-like protein 2 (SYTL2) and investigated the mechanisms regulating its expression and its gene function in OC. The mRNA expression of SYTL2 was significantly upregulated and the methylation of specific CpG sites within the SYTL2 promoter was decreased in the metastatic implants from the ovarian carcinoma xenografts compared to wild-type SK-OV-3 cells. Treatment with the demethylating agent 5-aza­2'-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A induced upregulation of SYTL2 in SK-OV-3 cells, implying that a DNA methylation-dependent epigenetic mechanism is involved in the regulation of SYTL2 expression. We also found that overexpression of SYTL2 promoted metastatic potential, including increased migration and invasiveness in the ovarian carcinoma cells. Furthermore, we utilized publicly available gene expression data to confirm the correlation between SYTL2 expression and poor prognosis in serous-type OC patients. Our findings provide novel evidence for the direct association of SYTL2 with the metastatic potential of ovarian carcinoma cells and its influence on metastatic recurrence of OC.

DNA hypomethylation-mediated overexpression of carbonic anhydrase 9 induces an aggressive phenotype in ovarian cancer cells.

PURPOSE: Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis. MATERIALS AND METHODS: We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line. RESULTS: Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells. CONCLUSION: Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells.

Overexpression of mucin 13 due to promoter methylation promotes aggressive behavior in ovarian cancer cells.

PURPOSE: Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies. MATERIALS AND METHODS: We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays. RESULTS: MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer. CONCLUSION: We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer.

Aberrant hypomethylation-mediated AGR2 overexpression induces an aggressive phenotype in ovarian cancer cells.

The metastatic properties of cancer cells result from genetic and epigenetic alterations that lead to the abnormal expression of key genes regulating tumor phenotypes. Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties; however, the precise regulatory mechanisms controlling metastasis-associated genes and their roles in metastatic transformation are largely unknown. We injected SK-OV-3 human ovarian cancer cells into the perineum of nude mice to generate a mouse model that mimics human ovarian cancer metastasis. We analyzed the mRNA expression and DNA methylation profiles in metastasized tumor tissues in the mice. The pro-oncogenic anterior gradient 2 (AGR2) gene showed increased mRNA expression and hypomethylation at CpG sites in its promoter region in the metastatic tumor tissues compared with the cultured SK-OV-3 cells. We identified crucial cytosine residues at CpG sites in the AGR2 promoter region. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced the level of CpG methylation in the AGR2 promoter and increased the level of AGR2 expression. Next, we explored the functional role of AGR2 in the metastatic transformation of SK-OV-3 cells. SK-OV-3 cells overexpressing AGR2 showed increased migratory and invasive activity. Our results indicate that DNA methylation within the AGR2 promoter modulates more aggressive cancer cell phenotypes.

Aberrant DNA methylation in the IFITM1 promoter enhances the metastatic phenotype in an intraperitoneal xenograft model of human ovarian cancer.

A lack of reliable biomarkers for the early detection and risk of metastatic recurrences makes ovarian cancer the most lethal gynecological cancer. To understand the molecular mechanisms involved in ovarian cancer metastasis in vivo, we analyzed the transcriptional expression pattern in metastatic implants of human ovarian carcinoma xenografts in mice. The expression of 937 genes was significantly different, by at least 2-fold, in the xenografts compared with that in SK-OV-3 cells. We investigated the mechanisms that regulate the expression of one of the profoundly upregulated genes, interferon-induced transmembrane protein 1 (IFITM1), in the metastatic implants. Specific CpG sites within the IFITM1 promoter were hypomethylated in the metastatic implants relative to those in the wild-type SK-OV-3 cells. Treating wild-type SK-OV-3 cells with the demethylating agent 5-aza-2'-deoxycytidine enhanced IFITM1 expression in a dose-dependent manner, implying transcriptional regulation by promoter methylation. We also found that IFITM1 overexpression caused increased migration and invasiveness in SK-OV-3 cells. Our results demonstrate that IFITM1 could be a novel metastasis-promoting gene that enhances the metastatic phenotype in ovarian cancer via epigenetic transcriptional regulation. Our findings also suggest that the status of DNA methylation within the IFITM1 promoter region could be a biomarker indicating metastatic progression in ovarian cancer.

Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line.

The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.

Dcr3 inhibit p53-dependent apoptosis in gamma-irradiated lung cancer cells.

PURPOSE: To identify genes responsible for the radiosensitivity, we investigated the role of the differential gene expression profiles by comparing radioresistant H1299 with radiosensitive H460 lung cancer cell lines. MATERIALS AND METHODS: mRNA profiles of lung cancer cell lines were assessed using microarray, and subsequent validation was performed with qRT-PCR (Quantitative real time-polymerase chain reaction). The expression levels of differentially expressed genes were determined by Western blot and the radioresistance of lung cancer cell lines was measured by clonogenic assay. RESULTS: From the differentially expressed apoptosis-related genes between H1299 and H460, we found Dcr3 (Decoy receptor 3, also known as TNFRSF6B; Tumour necrosis factor receptor super family member 6B) expression was significantly (P = 4.38 x 10(-7)) higher in H1299 cells than H460 cells. Moreover, the Dcr3 mRNA expression level in the radioresistant cell lines (H1299, A549, DLD1, MB231, MB157) was increased in comparison to the radiosensitive cell lines (ME180, Caski, U87MG, MCF7, H460). Overexpression of Dcr3 increased the survival rate of radiosensitive H460, MCF7, and U87MG cells, and knockdown of Dcr3 abolished the radioresistance of A549 cells. The survival rate of p53 (Tumour protein 53)-deficient H1299 after gamma-irradiation was not affected by the suppression of Dcr3 expression. However, when we introduced p53 into H1299 cells, siDcr3 (siRNA of Dcr3) suppressed the radioresistance of H1299 cells by inducing p53-dependent Fas (Fas receptor, also known as TNFRSF6; Tumour necrosis factor receptor super family member 6)-mediated apoptosis pathway. CONCLUSION: Characterisation of gene expression profiles in two lung cancer cell lines revealed that Dcr3 expression and p53-dependent apoptosis signalling pathway regulate cellular response to ionising radiation.

Tribbles-1 and -2 are tumour suppressors, down-regulated in human acute myeloid leukaemia.

Constitutive MAPK signalling is observed in approximately 50% of acute myeloid leukaemia (AML) cases. JNK activation in particular is associated with treatment failure in AML. Tribbles proteins (trb-1, trb-2 and trb-3) are potent negative regulators of MAPK pathways influencing apoptosis, differentiation and cell-cycle progression. Here we aimed to examine tribbles gene expression in AML and to characterise their role in leukaemic cells. A microarray dataset was interrogated for tribbles expression levels in AML cases and healthy controls. Myeloid cell proliferation and apoptosis were assayed in response to trb-1/trb-2 gene knockdown and overexpression, as well as a physical and functional interaction between trb and C/EBPalpha. Trb-2 expression was reduced in AML compared to healthy controls (correlating with nucleophosmin (NPM1) mutations), while low trb-1 expression was associated with inactive C/EBPalpha. In vitro assays indicated that trb-1/trb-2 are growth restrictive and pro-apoptotic in Me-1 cells, each capable of inhibiting JNK activation. JNK inactivation was itself associated with reduced Bcl-2 Ser70 phosphorylation, a residue which, when phosphorylated, maintains the anti-apoptotic activity of Bcl-2. Consistent with this, tribbles-mediated dephosphorylation of Bcl-2 Ser70 was associated with subsequent apoptosis. Trb-1/trb-2 transcription appeared to be moderately C/EBPalpha-responsive, and physical interaction between C/EBPalpha and trb-1/trb-2 was observed, suggesting a potential for auto-regulation of trb-1 and trb-2 transcription. In conclusion, we propose that trb-1 and trb-2 tumour suppressor activity may be abrogated in a proportion of AML patients. This may lead to enhanced cell survival, and therefore contribute to pathogenesis of the disease. Trb-1/trb-2 may, therefore, represent useful therapeutic targets for the treatment of AML in patients with dys-regulated trb activity.

Chemical inhibitors destabilize HuR binding to the AU-rich element of TNF-alpha mRNA.

Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3UTR to stabilize TNF-alpha mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-alpha mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC(50)) below 10 microM. The IC(50) of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 microM, respectively, for binding of HuR protein to TNF-alpha mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-alpha mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-alpha mRNA and decreased levels of secreted TNF-alpha. From these results, we could find inhibitors for the TNF-alpha mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs.

Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via MAPK signaling pathways.

Migration and proliferation of smooth muscle cells are key to a number of physiological and pathological processes, including wound healing and the narrowing of the vessel wall. Previous work has shown links between inflammatory stimuli and vascular smooth muscle cell proliferation and migration through mitogen-activated protein kinase (MAPK) activation, although the molecular mechanisms of this process are poorly understood. Here we report that tribbles-1, a recently described modulator of MAPK activation, controls vascular smooth muscle cell proliferation and chemotaxis via the Jun kinase pathway. Our findings demonstrate that this regulation takes place via direct interactions between tribbles-1 and MKK4/SEK1, a Jun activator kinase. The activity of this kinase is dependent on tribbles-1 levels, whereas the activation and the expression of MKK4/SEK1 are not. In addition, tribbles-1 expression is elevated in human atherosclerotic arteries when compared with non-atherosclerotic controls, suggesting that this protein may play a role in disease in vivo. In summary, the data presented here suggest an important regulatory role for trb-1 in vascular smooth muscle cell biology.