Development of a Real-Time qPCR Assay for Detection of Common MPL Mutations in Myeloproliferative Neoplasms (MPNS).

Myeloproliferative neoplasms (MPNs) are blood cell disorders, characterized by overproduction of abnormal cells in bone marrow due to stem cell mutation. The proliferations of blood cell are controlled by many genes particularly MPL gene which encodes thrombopoietin receptor, a hematopoietic growth factor involved in the production and regulation of the platelets and multipotent hematopoietic progenitor cells. Acquired mutations including (W515L and W515K) in this gene have been observed in patients with primary myelofibrosis or essential thrombocythemia lacking JAK2 (V617F) mutations. MPL mutation detection is important for MPNs diagnosis, but due to low frequency of mutant allele burden (< 15%) may be missed by already available common assays such as Sanger sequencing. Furthermore, these techniques are costly, time-consuming, and less sensitive. In present study, we aimed to develop sensitive, less time-consuming, and cost-effective real-time PCR assay for the detection of MPL mutations that is based on TaqMan fluorescent probes. DNA was extracted from blood sample of 128 MPNs patients collected and further analysis was performed on TaqMan RT-PCR. Reference curve was obtained for amplified product of MPL gene containing mutated sequence. The predicted sensitivity level was at least 5% mutant allele burden by our developed assay that is much higher than sequencing output. Out of 128, 2 (1.56%) patients harbored W515L mutation and 1 (0.78%) harbored W515K mutation. It was concluded that TaqMan qRT-PCR assay is an efficient, sensitive, cost-effective, and less time-consuming method capable of detecting MPL mutation in MPNs patients. We suggested that this assay might be helpful in investigating mutant allele load in MPNs patients.

L3MBTL2-mediated CGA transcriptional suppression promotes pancreatic cancer progression through modulating autophagy.

L3MBTL2 is a crucial component of ncPRC1.6 and has been implicated in transcriptional repression and chromatin compaction. However, the repression mechanism of L3MBTL2 and its biological functions are largely undefined. Here, we found that L3MBTL2 plays a distinct oncogenic role in tumor development. We demonstrated that L3MBTL2 repressed downstream CGA through an H2AK119ub1-dependent mechanism. Importantly, the binding of the MGA/MAX heterodimer to the E-box on the CGA promoter enhanced the specific selective repression of CGA by L3MBTL2. CGA encodes the alpha subunit of glycoprotein hormones; however, we showed that CGA plays an individual tumor suppressor role in PDAC. Moreover, CGA-transcript1 (T1) was identified as the major transcript, and the tumor suppression function of CGA-T1 depends on its own glycosylation. Furthermore, glycosylated CGA-T1 inhibited PDAC, partly by repression of autophagy through multiple pathways, including PI3K/Akt/mTOR and TP53INP2 pathways. These findings reveal the important roles of L3MBTL2 and CGA in tumor development.

TSPAN1-elevated FAM110A promotes pancreatic cancer progression by transcriptionally regulating HIST1H2BK.

Background: FAM110A belongs to the FAM110 family, which mainly functions in biological processes associated with the cell cycle. However, the biological functions in which FAM110A participates are largely undefined. In particular, its potential role in cancer remains unknown. The goal of this study was to uncover the role and mechanism of FAM110A in pancreatic cancer. Methods: Based on bioinformatics databases, qPCR and Western blot assays, we verified the elevated expression level of FAM110A in PDAC. Subsequently, FAM110A, HIST1H2BK and TSPAN1 overexpression or knockdown stable transfected cells were employed for biological functions' studies to explore the role in PDAC in vitro and in vivo. RNA-Seq, Western blot and luciferase-reporter assays were used to explore mechanism of FAM110A action in PDAC, and the involved pathway was verified by tumor phenotypic rescue experiments. Results: In this study, we demonstrated for the first time that FAM110A is an oncogene that promotes cell proliferation, migration, invasion and tumorigenesis in pancreatic cancer. HIST1H2BK was identified as the downstream target of FAM110A, while the promotion effect caused by FAM110A overexpression could be abolished by HIST1H2BK knockdown. Moreover, for the first time, we revealed the oncogenic role of HIST1H2BK in pancreatic cancer, and the tumor-promoting capacity of HIST1H2BK may be associated with its regulatory effect on G9a. In addition, we demonstrated that TSPAN1 displayed a positive transcriptional regulatory effect on FAM110A. Conclusions: Collectively, FAM110A plays an oncogenic role in PDAC, and the newly identified TSPAN1/FAM110A/HIST1H2BK/G9a pathway is involved in the modulation of pancreatic cancer progression and provides a novel prognostic and therapeutic strategy for pancreatic cancer treatment.

WBSCR22 and TRMT112 synergistically suppress cell proliferation, invasion and tumorigenesis in pancreatic cancer via transcriptional regulation of ISG15.

Pancreatic cancer (PC) is one of the most aggressive and devastating types of cancer owing to its poor prognosis and deadly characteristics. It is well established that aberrations in the expression of key regulatory genes, namely tumor suppressors and oncogenes, predispose patients to progression and metastasis of PC. Upregulation of Williams­Beuren syndrome chromosomal region 22 (WBSCR22) expression, a ribosomal biogenesis factor, has been reported in multiple types of human cancer. However, the role of WBSCR22 and its underlying mechanism in PC have not been well investigated. In the present study, the tumor suppressive role of WBSCR22 was reported in PC for the first time; the results indicated that WBSCR22 overexpression (OE) significantly suppressed cellular proliferation, migration, invasion and tumorigenesis in vivo and in vitro. RNA­sequencing analysis revealed that WBSCR22 negatively regulated the transcription of interferon­stimulated gene 15 (ISG15) downstream, which is a ubiquitin­like modifier protein involved in metabolic and proteasome degradation pathways, while the antitumor function of WBSCR22­OE could be rescued by ISG15 OE. In addition, the oncogenic role of ISG15 was further confirmed in PC; its upregulation promoted the proliferation, migration, invasion and tumorigenesis of PC. Furthermore, WBSCR22 and its cofactor tRNA methyltransferase activator subunit 11­2 (TRMT112) functioned synergistically in PC, and concurrent ectopic OE of WBSCR22 and TRMT112 further promoted the tumor suppressive potential of WBSCR22 in PC. Collectively, the findings indicated that WBSCR22 played an important role in PC development and that the WBSCR22/ISG15 axis may provide a novel therapeutic strategy for PC treatment.

Glioblastoma Therapy: Rationale for a Mesenchymal Stem Cell-based Vehicle to Carry Recombinant Viruses.

Evasion of growth suppression is among the prominent hallmarks of cancer. Phosphatase and tensin homolog (PTEN) and p53 tumor-suppressive pathways are compromised in most human cancers, including glioblastoma (GB). Hence, these signaling pathways are an ideal point of focus for novel cancer therapeutics. Recombinant viruses can selectivity kill cancer cells and carry therapeutic genes to tumors. Specifically, oncolytic viruses (OV) have been successfully employed for gene delivery in GB animal models and showed potential to neutralize immunosuppression at the tumor site. However, the associated systemic immunogenicity, inefficient transduction of GB cells, and inadequate distribution to metastatic tumors have been the major bottlenecks in clinical studies. Mesenchymal stem cells (MSCs), with tumor-tropic properties and immune privilege, can improve OVs targeting. Remarkably, combining the two approaches can address their individual issues. Herein, we summarize findings to advocate the reactivation of tumor suppressors p53 and PTEN in GB treatment and use MSCs as a "Trojan horse" to carry oncolytic viral cargo to disseminated tumor beds. The integration of MSCs and OVs can emerge as the new paradigm in cancer treatment.

Ribosome 18S m6A methyltransferase METTL5 promotes pancreatic cancer progression by modulating c­Myc translation.

Methyltransferase N6­adenosine (METTL5) is a methyltransferase that specifically catalyzes 18S rRNA N6 methylation at adenosine 1832 (m6A1832), which is located in a critical position in the decoding center, therefore suggesting its potential importance in the regulation of translation. However, the underlying mechanism of METTL5­mediated translation regulation of specific genes and its biological functions are largely undefined. To the best of our knowledge, the present study demonstrated for the first time that METTL5 was an oncogene that promoted cell proliferation, migration, invasion and tumorigenesis in pancreatic cancer. In addition, the oncogenic function of METTL5 may involve an increase in c­Myc translation, as evidenced by the fact that the oncogenic effect caused by METTL5 overexpression could be abolished by c­Myc knockdown. Notably, m6A modifications at the 5' untranslated region (5'UTR) and coding DNA sequence region (near the 5'UTR) of c­Myc mRNA played a critical role in the specific translation regulation by METTL5. In addition, it was further demonstrated that METTL5 and its cofactor tRNA methyltransferase activator subunit 11­2 synergistically promote pancreatic cancer progression. These findings revealed important roles for METTL5 in the development of pancreatic cancer and present the METTL5/c­Myc axis as a novel therapeutic strategy for treatment.

An overview of genetic mutations and epigenetic signatures in the course of pancreatic cancer progression.

Pancreatic cancer (PC) is assumed to be an intimidating and deadly malignancy due to being the leading cause of cancer-led mortality, predominantly affecting males of older age. The overall (5 years) survival rate of PC is less than 9% and is anticipated to be aggravated in the future due to the lack of molecular acquaintance and diagnostic tools for its early detection. Multiple factors are involved in the course of PC development, including genetics, cigarette smoking, alcohol, family history, and aberrant epigenetic signatures of the epigenome. In this review, we will mainly focus on the genetic mutations and epigenetic signature of PC. Multiple tumor suppressor and oncogene mutations are involved in PC initiation, including K-RAS, p53, CDKN2A, and SMAD4. The mutational frequency of these genes ranges from 50 to 98% in PC. The nature of mutation diagnosis is mostly homozygous deletion, point mutation, and aberrant methylation. In addition to genetic modification, epigenetic alterations particularly aberrant hypermethylation and hypomethylation also predispose patients to PC. Hypermethylation is mostly involved in the downregulation of tumor suppressor genes and leads to PC, while multiple genes also represent a hypomethylation status in PC. Several renewable drugs and detection tools have been developed to cope with this aggressive malady, but all are futile, and surgical resection remains the only choice for prolonged survival if diagnosed before metastasis. However, the available therapeutic development is insufficient to cure PC. Therefore, novel approaches are a prerequisite to elucidating the genetic and epigenetic mechanisms underlying PC progression for healthier lifelong survival.

miR-216a-mediated upregulation of TSPAN1 contributes to pancreatic cancer progression via transcriptional regulation of ITGA2.

Pancreatic cancer (PC) is recognized as the most aggressive and deadliest malignancy because it has the highest mortality of all cancers in humans. Mutations in multiple tumor suppressors and oncogenes have been documented to be involved in pancreatic cancer progression and metastasis. The upregulation of tetraspanin 1 (TSPAN1), a transmembrane protein, has been reportedly observed in many human cancers. However, the role of TSPAN1 and its underlying molecular mechanisms in PC progression have not been fully elucidated. In this study, we validated the oncogenic role of TSPAN1 in PC, showing that TSPAN1 reinforces cell proliferation, migration, invasion and tumorigenesis. To investigate the upregulation of TSPAN1 in PC, we showed that miR-216a is the upstream negative regulator of TSPAN1 via direct binding to the TSPAN1 3'-untranslated region. Through RNA-Seq analysis, we for the first time revealed that TSPAN1 expression transcriptionally regulates ITGA2, which is involved in the actin cytoskeleton pathway. The stimulated cell proliferation and invasion initiated by TSPAN1 overexpression could be abolished by knockdown of ITGA2 in PC cells. Furthermore, TSPAN1 epigenetically regulates the expression of ITGA2 by modulating the levels of TET2 DNMT3B and DNMT1, resulting in hypomethylation of the CpG island of the ITGA2 promoter. In conclusion, the newly identified miR-216a/TSPAN1/ITGA2 axis is involved in the modulation of PC progression and represents a novel therapeutic strategy for future pancreatic cancer treatment.

Aloperine in combination with therapeutic adenoviral vector synergistically suppressed the growth of non-small cell lung cancer.

PURPOSE: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and ranked top in terms of incidence and mortality in men and women. Recently, improvements in treatment approaches for NSCLC have reported, but still, there is a need to devise innovative treatment strategies, especially to manage the advanced and metastatic stage of NSCLC. Aloperine (ALO), an herbal alkaloid, has exerted anti-cancer effects in many cancers. However, the use of any chemotherapeutic agents is dose limited due to possible adverse effects and drug-resistance issues. Therefore, a combination of chemotherapy with viral-based targeted gene therapy may provide a novel treatment strategy for NSCLC. METHODS/RESULTS: In this study, the results of the MTT and flow cytometry-based assays showed that Aloperine-Adbic (adenoviral vector expressing p14ARF/p53) combined treatment on NSCLC cells synergistically produced anti-proliferative effects, induced apoptosis, and arrested cell cycle at the G1 phase. Furthermore, the expression analysis suggested that the p53/p21 pathway might contribute to achieving aforesaid cytotoxic effects. The ALO-Adbic combined treatment prolonged the percent survival of NSCLC xenograft models. CONCLUSION: In conclusion, ALO-Adbic combination can produce synergistic anti-cancer effects at low doses, and may offer a more effective and less toxic new treatment strategy for NSCLC.

Mesenchymal stem cell-mediated delivery of therapeutic adenoviral vectors to prostate cancer.

BACKGROUND: There is an urgent need for targeted biological therapies for prostate cancer with greater efficacy and less toxicity, particularly for metastatic disease, where current therapies are not curative. Therapeutic adenoviral vectors or oncolytic adenoviruses offer the possibility of a competent, nontoxic therapeutic alternative for prostate cancer. However, free viral particles must be delivered locally, an approach that does not address metastatic disease, and they display poor tumor penetration. To fully exploit the potential of these vectors, we must develop methods that improve intratumoral dissemination and allow for systemic delivery. This study establishes a proof-of-principle rationale for a novel human mesenchymal stem (stromal) cell-based approach to improving vector delivery to tumors. METHODS/RESULTS: We have generated mesenchymal stem cell-derived packaging cells for adenoviruses (E1-modified mesenchymal stem cells) by modifying human mesenchymal stem cells with the adenovirus (type C) E1A/B genes needed for viral replication. Using cell-based assays, we have demonstrated that two adenoviral vectors, replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus, packaged by E1A/B-modified mesenchymal stem cells, suppress the growth of prostate cancer cells in culture. Using subcutaneous xenograft models for human prostate cancer in mice, we have shown that E1A/B-modified mesenchymal stem cells display tumor tropism in tumor-bearing nude mice, that E1A/B-modified mesenchymal stem cells disseminate well within tumors, and that replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus-loaded E1-modified mesenchymal stem cells suppresses tumor growth in mice. CONCLUSION: The results show that this approach, if optimized, could circumvent the obstacles to efficient gene delivery encountered with current gene delivery approaches and provide an effective, nontoxic therapeutic alternative for metastatic disease.

A tumor targeting oncolytic adenovirus can improve therapeutic outcomes in chemotherapy resistant metastatic human breast carcinoma.

Breast cancer is the most prevalent malignancy in women, which remains untreatable once metastatic. The treatment of advanced breast cancer is restricted due to chemotherapy resistance. We previously investigated anti-cancer potential of a tumor selective oncolytic adenovirus along with cisplatin in three lung cancer cells; A549, H292, and H661, and found it very efficient. To our surprise, this virotherapy showed remarkable cytotoxicity to chemo-resistant cancer cells. Here, we extended our investigation by using two breast cancer cells and their resistant sublines to further validate CRAd's anti-resistance properties. Results of in vitro and in vivo analyses recapitulated the similar anti-tumor potential of CRAd. Based on the molecular analysis through qPCR and western blotting, we suggest upregulation of coxsackievirus-adenovirus receptor (CAR) as a selective vulnerability of chemotherapy-resistant tumors. CAR knockdown and overexpression experiments established its important involvement in the success of CRAd-induced tumor inhibition. Additionally, through transwell migration assay we demonstrate that CRAd might have anti-metastatic properties. Mechanistic analysis show that CRAd pre-treatment could reverse epithelial to mesenchymal transition in breast cancer cells, which needs further verification. These insights may prove to be a timely opportunity for the application of CRAd in recurrent drug-resistant cancers.

Cisplatin Synergistically Enhances Antitumor Potency of Conditionally Replicating Adenovirus via p53 Dependent or Independent Pathways in Human Lung Carcinoma.

Cisplatin is ranked as one of the most powerful and commonly prescribed anti-tumor chemotherapeutic agents which improve survival in many solid tumors including non-small cell lung cancer. However, the treatment of advanced lung cancer is restricted due to chemotherapy resistance. Here, we developed and investigated survivin promoter regulating conditionally replicating adenovirus (CRAd) for its anti-tumor potential alone or in combination with cisplatin in two lung cancer cells, H23, H2126, and their resistant cells, H23/CPR, H2126/CPR. To measure the expression of genes which regulate resistance, adenoviral transduction, metastasis, and apoptosis in cancer cells, RT-PCR and Western blotting were performed. The anti-tumor efficacy of the treatments was evaluated through flow cytometry, MTT and transwell assays. This study demonstrated that co-treatment with cisplatin and CRAd exerts synergistic anti-tumor effects on chemotherapy sensitive lung cancer cells and monotherapy of CRAd could be a practical approach to deal with chemotherapy resistance. Combined treatment induced stronger apoptosis by suppressing the anti-apoptotic molecule Bcl-2, and reversed epithelial to mesenchymal transition. In conclusion, cisplatin synergistically increased the tumor-killing of CRAd by (1) increasing CRAd transduction via enhanced CAR expression and (2) increasing p53 dependent or independent apoptosis of lung cancer cell lines. Also, CRAd alone proved to be a very efficient anti-tumor agent in cancer cells resistant to cisplatin owing to upregulated CAR levels. In an exciting outcome, we have revealed novel therapeutic opportunities to exploit intrinsic and acquired resistance to enhance the therapeutic index of anti-tumor treatment in lung cancer.

Knowledge, attitudes and practices survey on organ donation among a selected adult population of Pakistan.

BACKGROUND: To determine the knowledge, attitudes and practices regarding organ donation in a selected adult population in Pakistan. METHODS: Convenience sampling was used to generate a sample of 440; 408 interviews were successfully completed and used for analysis. Data collection was carried out via a face to face interview based on a pre-tested questionnaire in selected public areas of Karachi, Pakistan. Data was analyzed using SPSS v.15 and associations were tested using the Pearson's Chi square test. Multiple logistic regression was used to find independent predictors of knowledge status and motivation of organ donation. RESULTS: Knowledge about organ donation was significantly associated with education (p = 0.000) and socioeconomic status (p = 0.038). 70/198 (35.3%) people expressed a high motivation to donate. Allowance of organ donation in religion was significantly associated with the motivation to donate (p = 0.000). Multiple logistic regression analysis revealed that higher level of education and higher socioeconomic status were significant (p < 0.05) independent predictors of knowledge status of organ donation. For motivation, multiple logistic regression revealed that higher socioeconomic status, adequate knowledge score and belief that organ donation is allowed in religion were significant (p < 0.05) independent predictors. Television emerged as the major source of information. Only 3.5% had themselves donated an organ; with only one person being an actual kidney donor. CONCLUSION: Better knowledge may ultimately translate into the act of donation. Effective measures should be taken to educate people with relevant information with the involvement of media, doctors and religious scholars.