Transforming Growth Factor and the Role of Epigenetic Aberrancies in Oncogenic Amplifications: A New Perspective in Preventive and Therapeutic Arena

Three genetic mechanisms activate oncogenes in human neoplasms: 1) mutations, 2) gene amplification, and 3) chromosome rearrangements. These mechanisms result in either an alteration of protooncogene structure or an increase in protooncogene expression. The role of epigenetic aberrancies in carcinogenesis has been described earlier however to clinicians, the biological implications of epigenetic therapies to prevent cancer and the mechanisms involved have been a mystery. Furthermore, there is no biomarker suggested to track the carcinogenesis steps long before cancer develops, and this has caused a significant lack of proactive and preventive measures to be taken as all recommendations in preventive oncology are either deficiently and blindly made or through screening methods which are too late in the game. Here we explored a very different approach by applying our deepest understanding of epigenetics and carcinogenesis and even further we developed a framework where our clinical findings could translate to the research and vice versa by generating advanced and novel hypotheses on “how we get cancer”, by exploring the relation between the host and the tumor cells in a way no one had perceived before. The role of specific cancer stem cell pathways is dissected and how to inhibit each of these initiators using multitargeted epigenetic therapies and off-label medications are explained. We should admit that without considering this sophisticated amazing biological network, cancer will remain an unsolved challenge. Further, we were able to solve this unsolved puzzle by bridging the gap from a hypothetical point of view/hypothesis to possibilities that explain the clinical findings we had observed, and conclude that such an approach can completely change the way practitioners are treating cancer.

The Frequency of the v-AKT Murine Thymoma Viral Oncogene Homologue 1 Gene Amplification among Sudanese Women with Ovarian Cancer: A Cross-Sectional Study

Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to the AKT family among Sudanese women with OC. The present study was conducted to detect the AKT1 gene amplification and its association with tumour types, grades, and ages among Sudanese women with OC, bearing in mind the ethnic variation. Methods: This institution-based study included 79 cases of women diagnosed with ovarian cancer (OC) at Omdurman Maternity Hospital in the period 2013-2018. Formalin-fixed, paraffin-embedded (FFPE) tissue sections were used to extract RNA. AKT1 gene amplification was assessed using quantitative real-time PCR. Results: The mean age (±SD) of included women was 49.29 (±13.612). The amplification of AKT1 gene was observed in 18/79 (22.8%) of OC women, with a high frequency in women with undifferentiated 1/2 (50%), clear cell 2/6 (33.3%), mucinous 3/11 (27.3%), endometrioid 3/17 (17.6%), and serous carcinomas 5/30 OC (16.7%). High frequency was seen in women with low (26.3%;n = 10/28) rather than in higher (19.5%;n = 8/33) grade carcinoma, and in older (25.8%;n = 8/23) rather than younger (18.2%;n = 2/9) women. No significant association between AKT1 gene amplification and tumour types, grades, and ages of women was observed (Fisher’s Exact test: p = 0.405, 0.593 and 0.851, respectively). Conclusion: AKT1 gene amplification arises in around one-fifth of Sudanese women with ovarian cancer (OC). It is seen more in undifferentiated, clear cell, and mucinous tumours types, and more frequently in low tumour grade and older women, but not to a statistically significant level. These outcomes sustenance previous studies suggesting that activated AKT genes have a vital role in OC progression and may offer a plan for targeted therapy and prognostic evaluation.

Application of Abnormal Touchdown PCR with High Degeneracy Primer in Amplification of Large-Family Genes

[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.

Chromosomal Localization of Genes bz1,bz2 in Maize by Using Ultra-sensitive FISH with Tyramide Signal Amplification(TSA-FISH)

It has been reported that endosperm undergoes programmed cell death (PCD) during maize kernel development.Both bz1 (bronze ) and bz2 are anthocyanin biosynthetic genes,and related to development of aleuronic layer of maize seeds.Tyramide signal amplification fluorescence in situ hybridization (TSA FISH) is a novel and high sensitive FISH technique,which is suitable for routine application in plant cytogenetic research.Using this technique,we physically mapped the bz1 gene onto the short arm of chromosome 9 and the long arm of chromosome 1;the percentage distances from centromere to hybridization site were 40.2,75.4 respectively,and the bz2 onto the long arm of chromosome 1 and the short arm of chromosome 5;the percentage distances from centromere to hybridization site were 21.6,15.3 separately.The TSA FISH techniques of small low copy DNA sequences for plants are discussed.

Study of differential polymerase chain reaction of C-erbB-2 oncogene amplification in gastric cancer

AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P＜0.05) and surgical specimens with lymph node metastasis (P＜0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P＜0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P＜0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P＜0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.

Estrogen receptor alpha gene amplification in breast cancer:25 years of debate

Twenty-five years ago,Nembrot and colleagues reported amplification of the estrogen receptor alpha gene(ESR1) in breast cancer,initiating a broad and still ongoing scientific debate on the prevalence and clinical significance of this genetic aberration,which affects one of the most important genes in breast cancer.Since then,a multitude of studies on this topic has been published,covering a wide range of divergent results and arguments.The reported prevalence of this alteration in breast cancer ranges from 0% to 75%,suggesting that ESR1 copy number analysis is hampered by technical and interpreter issues.To date,two major issues related to ESR1 amplification remain to be conclusively addressed:(1) The extent to which abundant amounts of messenger RNA can mimic amplification in standard fluorescence in situ hybridization assays in the analysis of strongly expressed genes like ESR1,and(2) the clinical relevance of ESR1 amplification:Such relevance is strongly disputed,with data showing predictive value for response as well as for resistance of the cancer to anti-estrogen therapies,or for subsequent development of cancers in the case of precursor lesions that display amplification of ESR1.This review provides a comprehensive summary of the various views on ESR1 amplification,and highlights explanations for the contradictions and conflicting data that could inform future ESR1 research.

ANALYSIS OF C-HA-RAS GENE AMPLIFICATION AND MUTATION IN LARYNGEAL CARCINOMA

In order to study the altered molecular events during laryngeal carcinogenesis and elucidate the role of Haras oncogene amplification and mutation. we have examined their profile by polymerase chain reaction (PCR) and selective oligonucleotide hybridization. We analyzed the mutational status of codon 12 of Haras in 22 laryngeal carcinomas and 10 normal tissues. and found that 7 of 22 laryngeal carcinomas contained a Ha-ras mutation at codon 12. The frequency of mutation was 32%. None of the normal tissues revealed mutation. Moreover. no amplification was found in cancers when compared to the normal. Ourfindings indicated that the activated Ha-ras gene existed in laryngeal carcinoma. and activation of the Haras gene by mutation at codon 12 might play a key role in laryngeal carcinogenesis.

Rapid,specific and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification targeted to vvhA gene

Vibrio vulnificus is an estuarine bacterial pathogen for human.The rapid,specific and sensitive detection of V.vulnificus is urgently needed for early disease diagnosis and timely treatment of V.vulnificus infection.In the study,a loop-mediated isothermal amplification（LAMP） technique was developed for V.vulnificus detection with a set of primers,composed of two out primers and two inner primers targeted to vvh A gene.The optimal amplification temperature was 63°C and the reaction only took 35 min.The amplification products could not only be detected by agarose gel electrophoresis with ladder-like pattern bands,but also could be visualized using calcein with naked eye directly.Forty-five strains were tested for the specificity of LAMP assay,and all the V.vulnificus strains were identified correctly while other strains were negative results.The sensitive of the new LAMP assay was 100-fold more sensitive than the conventional PCR.Meanwhile,all the V.vulnificus strains were detected correctly in spiked,clinical and environmental samples by the new LAMP assay.Compared with other well-known techniques,the new LAMP assay targeted to vvh A gene was extremely rapid,simple,sensitive and specific for V.vulnificus identification.

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.

INDUCTION OF C-MYC GENE AMPLIFICATION BY HYDROXYUREA AND ITS INHIBITION BY HOMOHARRINGTONINE

Induction of c-myc gene amplification in L1210 cells by hydroxyurea and its inhibition by homohar-ringtonine were investigated using the DNA-DNA molecular hybridization technique. When the cells were treated with hydroxyurea 1.0 mM for 16 hours, and incubated a further 16 hours in a drug-free medium, the c-myc gene amplified 23.5-fold. If homohar-ringtonine 50 μM was used at the same time as hydroxyurea, gene amplification did not occur. Cycloheximide, an inhibitor of protein biosynthesis, produced a similar effect. Our results indicated that a (or some) protein factor(s) might be involved in gene amplification. Detailed analysis showed that the synthesis of this protein factor(s) started 4 hours before the initiation of the S phase but did not continue in the S phase. It was also found that this protein factor(s) was very labile and began to degrade 2 hours after its appearance.

Progress in clinical diagnosis and treatment of colorectal cancer with rare genetic variants

Targeted therapy is crucial for advanced colorectal cancer(CRC) positive for genetic drivers. With advances in deep sequencing technology and new targeted drugs, existing standard molecular pathological detection systems and therapeutic strategies can no longer meet the requirements for careful management of patients with advanced CRC. Thus, rare genetic variations require diagnosis and targeted therapy in clinical practice. Rare gene mutations, amplifications, and rearrangements are usually associated with poor prognosis and poor response to conventional therapy. This review summarizes the clinical diagnosis and treatment of rare genetic variations, in genes including erb-b2 receptor tyrosine kinase 2(ERBB2), B-Raf proto-oncogene, serine/threonine kinase(BRAF), ALK receptor tyrosine kinase/ROS proto-oncogene 1, receptor tyrosine kinase(ALK/ROS1), neurotrophic receptor tyrosine kinases(NTRKs), ret proto-oncogene(RET), fibroblast growth factor receptor 2(FGFR2), and epidermal growth factor receptor(EGFR), to enhance understanding and identify more accurate personalized treatments for patients with rare genetic variations.

Cloning and Bioinformatics Analysis of hcp Gene in Aeromonas hydrophila

[Objectives]To explore the function of hcp gene in Aeromonas hydrophila.[Methods]A pair of specific primers was designed referring to the hcp gene sequence of A.hydrophila.The hcp gene was amplified by PCR,and performed bioinformatics analysis.[Results]The hcp gene had a total length of 1650 bp and encoded 549 amino acids.The theoretical molecular weight of the protein predicted was about 59476.44 kDa.After predicting the N-terminal signal peptide structure of the amino acid sequence,neither obvious signal peptide cleavage site nor signal peptide was found,and the protein had no transmembrane region.The amino acid sequence had a N-glycosylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,9 N-myristoylation sites,4 isoprene binding sites,10 microbody C-terminal target signal sites,and an ATP/GTP binding site motif A(P-ring).The amino acid sequence of hcp gene of A.hydrophila was performed homology analysis with other Aeromonas strains,and it showed higher homology with A.veronii.In the secondary structure,theα-helix,β-sheet,random coil and extended strand accounted for 45.36%,6.01%,37.52%and 11.11%,respectively.The tertiary structure model consisted of 18α-helix and 22β-sheet.Analysis of protein-protein network interaction demonstrated that the proteins interacting with Hcp protein were AHA_3407,nrfA,nirB-1,nirB-2 and AHA_1112.[Conclusions]Through the bioinformatics prediction results,the basic information of hcp gene of A.hydrophila is preliminarily understood,and the possible function of this protein is predicted,in order to provide guidance for subsequent vaccine research.

Molecular Cloning and Bioinformatics Analysis of sucC Gene of Vibrio alginolyticus Strain HY9901

[Objectives]To clone the sucC gene of Vibrio alginolyticus strain HY9901 and conduct the bioinformatics analysis.[Methods]Based on the sucC gene of V.alginolyticus strain HY9901,specific primers were designed to amplify the full length sequence by PCR and make further analysis.[Results]The theoretical molecular weight of SucC protein was about 41528.45 Da,and the full length was 1167 bp,encoding 388 amino acids.It has no signal peptide and transmembrane region,and has a variety of functional sites.It is predicted that it is mainly located in the cytoplasm,and the ubiquitin and lactate modification sites overlap,and it has high gene homology with Vibrio parahaemolyticus.Theα-helix,random coil and extended strand are the main secondary structures.The similarity between the constructed three-level structure model and the template is high.[Conclusions]This study reveals the structural characteristics and functional potential of SucC protein,and provides a theoretical basis for the study of drug resistance mechanism and prevention strategies.

Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay

When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.

Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.

西安地区256例Gene Xpert MTB/RIF阳性肺结核患者rpoB基因突变特征分析

目的分析西安地区256例利福平耐药实时荧光定量核酸扩增检测技术(Gene Xpert MTB/RIF)阳性肺结核患者rpoB基因突变特征。方法本研究为回顾性分析。256株结核分枝杆菌(MTB)菌株分离自2020年6月至2022年6月于陕西省结核病防治院就诊的Gene Xpert MTB/RIF阳性肺结核门诊及住院患者,菌株无重复收集,来自痰液标本174份、支气管肺泡灌洗液标本82份,患者年龄(45.67±8.36)岁。采用DNA直接测序法对256株利福平耐药MTB菌株rpoB基因的PCR产物进行分析。将256株利福平耐药MTB菌株根据利福平耐药程度分为低、中、高耐药MTB菌株,采用χ^(2)检验比较3种菌株突变位点。人工诱导3株利福平耐药MTB菌株,采用DNA直接测序法对其rpoB基因的PCR产物进行分析。结果测序报告显示,256株利福平耐药MTB菌株中有253株发生rpoB基因位点突变,突变率为98.83%(253/256)。突变类型包括C→T、T→G、C→G、A→T、C→A、A→G、G→A、G→T、T→C、A→C共计10种,涉及丝氨酸、亮氨酸、丙氨酸、组氨酸、酪氨酸、谷氨酸、赖氨酸、精氨酸、天冬氨酸、脯氨酸、蛋氨酸、缬氨酸、异亮氨酸、甘氨酸共14个氨基酸密码子,均为点突变。利福平耐药菌株突变主要集中在531位[53.75%(136/253)]、526位[23.32%(59/253)],其他位点包括513、516、533、515、513、532、522、511、519、518、533。高耐药MTB菌株531位氨基酸突变发生率与低、中耐药MTB菌株比较[66.91%(91/136)比37.88%(25/66)、37.04%(20/54)],差异有统计学意义(χ^(2)=22.154,P<0.001);低、中耐药MTB菌株531位氨基酸突变发生率比较,差异无统计学意义(P>0.05)。低、中、高耐药MTB菌株526位氨基酸突变发生率比较[22.73%(15/66)、25.93%(14/54)、22.06%(30/136)],差异无统计学意义(χ^(2)=0.331,P=0.847)。人工诱导的3株利福平耐药MTB菌株均发生rpoB基因位点突变,低、中耐药MTB菌株突变均位于526位点,高耐药MTB菌株突变位于531位点。结论西安地区Gene Xpert MTB/RIF阳性肺结核患者rpoB基因突变率较高,以点突变为主,主要集中在531位、526位,531位TCG→TTG突变在rpoB基因突变类型中突变频率最高,且与高耐药有关。

Amplification and sequencing of a sulfur-rich 10kd prolamin gene from rice seeds

Nutritious value of seed storage protein is low due to deficiency in essential amino acid contents. Cereals are mainly deficient in lysine and legumes in sulfur-containing amino acids (methionine and cysteine). So far, several sufur-rich seed protein genes have been isolated and the essential amino acid contents of seed proteins were increased in transgenic tobacco and Brassica napus. In this paper we report the isolation and sequencing of the 10kd prolamin gene from seeds. Poly(A) RNA were prepared from the immature endosperms of japonica rice, Sachiminori, 10 d after flowering. Complementary DNAs were synthesized according to Promega Instruction Manual. Two primers were synthesized and their sequences were Primer Ⅰ: CGTCTACACCATCTGGAATC, Primer Ⅱ: GTGTTTGCAGATAGTATGC. The amplified fragraents were inserted into the Sma I site of pGEM-7zf(+) and was used to transform E. coli JM101 after PCR reaction. DNA sequence were determined by Sanger’s Chain-termination method. Synthesis of cDNA. Using mRNAs as

Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology.In comparison to plasmid-based multi-copy expression,the utilization of chromosomal multi-copy genes offers increased stability of expression level,diminishes the metabolic burden on host cells,and enhances overall genetic stability.In this study,we developed the“BacAmp”,a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis,which was achieved by employing a combination of repressor and non-natural amino acids(ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination.When the reversible switch is turned on,genome editing and gene amplification can be achieved.Subsequently,the reversible switch was turned off therefore stabilizing the gene copy number.The stabilized gene amplification system marked by green fluorescent protein,achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations.When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid(NeuAc)synthesis,the copy number of the critical gene increased to an average of 7.7,which yielded a 1.3-fold NeuAc titer.Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B.subtilis.

Evaluation of molecular residual disease in operable non-small cell lung cancer with gene fusions,MET exon skipping or de novo MET amplification

Gene fusions and MET alterations are rare and difficult to detect in plasma samples.The clinical detection efficacy of molecular residual disease(MRD)based on circulating tumor DNA(ctDNA)in patients with non-small cell lung cancer(NSCLC)with these mutations remains unknown.This prospective,non-intervention study recruited 49 patients with operable NSCLC with actionable gene fusions(ALK,ROS1,RET,and FGFR1),MET exon 14 skipping or de novo MET amplification.We analyzed 43 tumor tissues and 111 serial perioperative plasma samples using 1021-and 338-gene panels,respectively.Detectable MRD correlated with a significantly higher recurrence rate(P<0.001),yielding positive predictive values of 100%and 90.9%,and negative predictive values of 82.4%and 86.4%at landmark and longitudinal time points,respectively.Patients with detectable MRD showed reduced disease-free survival(DFS)compared to those with undetectable MRD(P<0.001).Patients who harbored tissue-derived fusion/MET alterations in their MRD had reduced DFS compared to those who did not(P=0.05).To our knowledge,this is the first comprehensive study on ctDNA-MRD clinical detection efficacy in operable NSCLC patients with gene fusions and MET alterations.Patients with detectable tissue-derived fusion/MET alterations in postoperative MRD had worse clinical outcomes.

The delivery of N-myc downstream-regulated gene 2(NDRG2)self-amplifying mRNA via modified lipid nanoparticles as a potential treatment for drug-resistant and metastatic cancers

The protein,N-myc downstream-regulated gene 2(NDRG2),a tumor suppressor,is significantly decreased or absent in many types of cancer.There is a significant negative correlation between the levels of NDRG2 and the development and progression of cancer tumor recurrence and tumor invasion,in different cancers.In contrast,the in vitro and in vivo overexpression of the NDRG2 protein decreases the proliferation,growth,adhesion and migration of many types of cancer cells.The in vitro overexpression of NDRG2 increases the efficacy of certain anticancer drugs in specific types of cancer cells.We hypothesize that the delivery of the mRNA of the NDRG2 protein,encapsulated by lipid nanoparticles,could represent a potential treatment of metastatic and drug-resistant cancers.This would be accomplished using a self-amplifying mRNA that encodes the NDRG2 protein and an RNA-dependent-RNA polymerase,obtained from an in vitrotranscribed(IVT)mRNA.The IVT mRNA would be encapsulated in a lipid nanoformulation.The efficacy of the nanoformulation would be determined in cultured cancer cells and if the results are positive,nude mice transplanted with either drug-resistant or metastatic drug-resistant cancer cells,would be treated with the nanoformulation and monitored for efficacy and adverse effects.If the appropriate preclinical studies indicate this formulation is efficacious and safe,it is possible it could be evaluated in clinical trials.