Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells

AbstractOur previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA) expression in the progression of meningioma. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Initially, radiation treatment was found to induce hypomethylation in meningioma cells with a decrease in DNA (cytosine-5)-methyltransferase 1 (DNMT1) and methyl-CpG binding domain protein (MBD) expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with N-acetyl cysteine (NAC) did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression, whereas pre-treatment with sodium butyrate inhibited radiation-induced uPA expression, which complemented our prior results. Methylation-specific polymerase chain reaction on bisulfite-treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells. Transfection with small hairpin RNA (shRNA)-expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decrease in invasion and proliferation of meningioma cells. Further, radiation treatment was found to recruit SP1 transcription factor, which was abrogated by shRNA treatment. Analysis on signaling events demonstrated the activation of MAP kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in radiation-treated cells, while U0126 (MEK/ERK inhibitor) blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors treated with radiation compared to untreated tumors. In conclusion, our data suggest that radiation-mediated hypomethylation triggers uPA expression in meningioma cells

Suppression of uPAR Retards Radiation-Induced Invasion and Migration Mediated by Integrin β1/FAK Signaling in Medulloblastoma

Despite effective radiotherapy for the initial stages of cancer, several studies have reported the recurrence of various cancers, including medulloblastoma. Here, we attempt to capitalize on the radiation-induced aggressive behavior of medulloblastoma cells by comparing the extracellular protease activity and the expression pattern of molecules, known to be involved in cell adhesion, migration and invasion, between non-irradiated and irradiated cells.We identified an increase in invasion and migration of irradiated compared to non-irradiated medulloblastoma cells. RT-PCR analysis confirmed increased expression of uPA, uPAR, focal adhesion kinase (FAK), N-Cadherin and integrin subunits (e.g., α3, α5 and β1) in irradiated cells. Furthermore, we noticed a ∼2-fold increase in tyrosine phosphorylation of FAK in irradiated cells. Immunoprecipitation studies confirmed increased interaction of integrin β1 and FAK in irradiated cells. In addition, our results show that overexpression of uPAR in cancer cells can mimic radiation-induced activation of FAK signaling. Moreover, by inhibiting FAK phosphorylation, we were able to reduce the radiation-induced invasiveness of the cancer cells. In this vein, we studied the effect of siRNA-mediated knockdown of uPAR on cell migration and adhesion in irradiated and non-irradiated medulloblastoma cells. Downregulation of uPAR reduced the radiation-induced adhesion, migration and invasion of the irradiated cells, primarily by inhibiting phosphorylation of FAK, Paxillin and Rac-1/Cdc42. As observed from the immunoprecipitation studies, uPAR knockdown reduced interaction among the focal adhesion molecules, such as FAK, Paxillin and p130Cas, which are known to play key roles in cancer metastasis. Pretreatment with uPAR shRNA expressing construct reduced uPAR and phospho FAK expression levels in pre-established medulloblastoma in nude mice.

ABHD11-AS1: An Emerging Long Non-Coding RNA (lncRNA) with Clinical Significance in Human Malignancies

The aberrant expression of lncRNAs has been linked to the development and progression of different cancers. One such lncRNA is ABHD11 antisense RNA 1 (ABHD11-AS1), which has recently gained attention for its significant role in human malignancies. ABHD11-AS1 is highly expressed in gastric, lung, breast, colorectal, thyroid, pancreas, ovary, endometrium, cervix, and bladder cancers. Several reports highlighted the clinical significance of ABHD11-AS1 in prognosis, diagnosis, prediction of cancer progression stage, and treatment response. Significantly, the levels of ABHD11-AS1 in gastric juice had been exhibited as a clinical biomarker for the assessment of gastric cancer, while its serum levels have prognostic potential in thyroid cancers. The ABHD11-AS1 has been reported to exert oncogenic effects by sponging different microRNAs (miRNAs), altering signaling pathways such as PI3K/Akt, epigenetic mechanisms, and N6-methyladenosine (m6A) RNA modification. In contrast, the mouse homolog of AHD11-AS1 (Abhd11os) overexpression had exhibited neuroprotective effects against mutant huntingtin-induced toxicity. Considering the emerging research reports, the authors attempted in this first review on ABHD11-AS1 to summarize and highlight its oncogenic potential and clinical significance in different human cancers. Lastly, we underlined the necessity for future mechanistic studies to unravel the role of ABHD11-AS1 in tumor development, prognosis, progression, and targeted therapeutic approaches

Apoptosis Induced by Knockdown of uPAR and MMP-9 is Mediated by Inactivation of EGFR/STAT3 Signaling in Medulloblastoma

BACKGROUND: Medulloblastoma is a highly invasive cancer of central nervous system diagnosed mainly in children. Matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator receptor (uPAR) are over expressed in several cancers and well established for their roles in tumor progression. The present study is aimed to determine the consequences of targeting these molecules on medulloblastoma progression. METHODOLOGY/PRINCIPAL FINDINGS: Radiation is one of the foremost methods applied for treating cancer and considerable evidence showed that radiation elevated uPAR and MMP-9 expression in medulloblastoma cell. Therefore efforts are made to target these molecules in non-irradiated and irradiated medulloblastoma cells. Our results showed that siRNA-mediated knockdown of uPAR and MMP-9, either alone or in combination with radiation modulated a series of events leading to apoptosis. Down regulation of uPAR and MMP-9 inhibited the expression of anti-apoptotic molecules like Bcl-2, Bcl-xL, survivin, XIAP and cIAPI; activated BID cleavage, enhanced the expression of Bak and translocated cyctochrome C to cytosol. Capsase-3 and -9 activities were also increased in uPAR- and MMP-9-downregulated cells. The apoptosis induced by targeting MMP-9 and uPAR was initiated by inhibiting epidermal growth factor receptor (EGFR) mediated activation of STAT3 and NF-κB related signaling molecules. Silencing uPAR and MMP-9 inhibited DNA binding activity of STAT3 and also reduced the recruitment of STAT3 protein at the promoter region of Bcl-2 and survivin genes. Our results suggest that inhibiting uPAR and MMP-9 reduced the expression of anti-apoptotic molecules by inactivating the transcriptional activity of STAT3. In addition, treating pre-established medulloblastoma with siRNAs against uPAR and MMP-9 both alone or in combination with radiation suppressed uPAR, MMP-9, EGFR, STAT3 expression and induced Bak activation leading to apoptosis. CONCLUSION/SIGNIFICANCE: Taken together, our results illustrated that RNAi mediated targeting of uPAR and MMP-9 might have therapeutic potential against medulloblastoma

Apoptosis Induced by Knockdown of uPAR and MMP-9 is Mediated by Inactivation of EGFR/STAT3 Signaling in Medulloblastoma

Background: Medulloblastoma is a highly invasive cancer of central nervous system diagnosed mainly in children. Matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator receptor (uPAR) are over expressed in several cancers and well established for their roles in tumor progression. The present study is aimed to determine the consequences of targeting these molecules on medulloblastoma progression. Methodology/Principal Findings: Radiation is one of the foremost methods applied for treating cancer and considerable evidence showed that radiation elevated uPAR and MMP-9 expression in medulloblastoma cell. Therefore efforts are made to target these molecules in non-irradiated and irradiated medulloblastoma cells. Our results showed that siRNA-mediated knockdown of uPAR and MMP-9, either alone or in combination with radiation modulated a series of events leading to apoptosis. Down regulation of uPAR and MMP-9 inhibited the expression of anti-apoptotic molecules like Bcl-2, Bcl-xL, survivin, XIAP and cIAPI; activated BID cleavage, enhanced the expression of Bak and translocated cyctochrome C to cytosol. Capsase-3 and -9 activities were also increased in uPAR- and MMP-9-downregulated cells. The apoptosis induced by targeting MMP-9 and uPAR was initiated by inhibiting epidermal growth factor receptor (EGFR) mediated activation of STAT3 and NF-kB related signaling molecules. Silencing uPAR and MMP-9 inhibited DNA binding activity of STAT3 and also reduced the recruitment of STAT3 protein at the promoter region of Bcl-2 and survivin genes. Our results suggest that inhibiting uPAR and MMP-9 reduced the expression of anti-apoptotic molecules by inactivating the transcriptional activity of STAT3. In addition, treating pre-established medulloblastoma with siRNAs against uPAR and MMP-9 both alone or in combination with radiation suppressed uPAR, MMP-9, EGFR, STAT3 expression and induced Bak activation leading to apoptosis. Conclusion/Significance: Taken together, our results illustrated that RNAi mediated targeting of uPAR and MMP-9 might have therapeutic potential against medulloblastoma

uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma.

BACKGROUND:Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model. CONCLUSION/SIGNIFICANCE:These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma

uPAR and MMP-9 activates EGFR and STAT3; Inhibition of STAT3 and NF-κB 65 induces apoptosis in Daoy and D283 cells.

<p>To determine possible cross-talk between STAT3 and Rel-A during apoptosis, each gene was downregulated using specific siRNA followed by determination of the nuclear levels of STAT3 and Rel-A in both Daoy and D283 cells. <b>A)</b> Medulloblastoma cells transfected with full-length uPAR expressing plasmid or/and incubated with recombinant MMP-9 (25 ηg/ml), were either blocked with EGFR neutralizing antibody or non-specific isotype IgG to determine the role of the extracellular proteases in activation of EGFR and STAT3. Western blot analysis was carried out on cell lysates isolated from Daoy and D283 to determine the levels of phosphorylated EGFR and STAT3. <b>B)</b> Western blot analysis was carried out on the nuclear extracts isolated from STAT3-downregulated cells. Membranes were probed with phosphorylated forms of STAT3 and Rel-A. <b>C)</b> Similarly, nuclear extracts isolated from Rel-A downregulated cells were evaluated by Western blotting to detect phosphorylated forms of Rel-A and STAT3 in the nucleus. <b>D)</b> Chromatin immunoprecipitation assay was carried out with the nuclear extracts isolated from Day and D283 cells transfected with either STAT3 siRNA or pUM plasmid. Chromatin was immunopreciptated with STAT3 and analyzed by PCR using primers specific for Bcl-2 promoter region to determine the STAT3 recruitment at Bcl-2 promoter sequences. Chromatin immunoprecipitated with isotype IgG was used as negative control. Input DNA was confirmed by amplifying the Bcl-2 promoter form the chromatin aliquot collected prior to immunoprecipitation step.</p

Transfection with pU, pM and pUM in combination with radiation specifically down regulates expression of uPAR and MMP-9 and induces apoptosis.

<p>Daoy and D283 cell lines were transfected with either transfection reagent alone (control, Con), pSV (scrambled vector) or gene specific shRNA’s as described in materials and methods. <b>A)</b> RT-PCR analysis and western blotting was carried to determine the expression levels of uPAR and MMP-9 in cell transfected with reagent, pSV, pU-, pM- and pUM-transfected Daoy and D283 cells (with and without radiation, 8 Gy). The experiments were repeated three times and representative images were shown. The immunoblots were stripped and re-probed with GAPDH as a loading control. Semi-quantitative RT-PCR analysis was carried out to detect mRNA levels of uPAR and MMP-9 using specific primers. <b>B)</b> Protein band and PCR amplicon intensities were quantified by densitometry analysis using ImageJ software (National Institutes of Health). The levels of uPAR and MMP-9 protein were normalized to GAPDH levels in mock-transfected cells. <i>Columns:</i> mean of triplicate experiments; <i>bars</i>: s.d.; *<i>p<</i>0.01, significant difference from pSV-transfected cells. <b>C)</b> 72 hrs after Transfection (with or without radiation), the cells were trypsinized and analyzed by flow cytometry to measure the number of apoptotic TUNEL-positive cells using the APO-BrdU TUNEL Assay kit. The percent apoptotic cells from each treatment are represented and the mean ± s.d. from three separate experiments was represented; *<i>p<</i>0.05 and ** <i>p</i><0.01 were considered significant compared to pSV-transfected cells and pSV+IR treated cells, respectively. <b>D)</b> Apoptotic cells were also quantified using an Annexin V assay followed by FACS analysis. Results are reported as the percent of cells (minimum 10,000 analyzed) that were Annexin V-positive. Gating was based on positive and negative control cells. Representative FACS data of Daoy and D283 are shown (<i>n</i> = 3).</p