A Novel Strategy To Inhibit Melanoma Metastasis Based On JWA

The incidence and mortality of melanoma have dramatically increased over the past several decades,which has become a major public health problem worldwide.Modern clinical practice has substantially altered the prognosis of melanoma due to early detection and improved therapeutic regimens;however,the much acclaimed success of targeted therapy only increases a few months of survival advantage to late-stage cancer patients.Metastasis is still accounted for over 90%of cancer related deaths.Therefore,the more effective anti-cancer strategy 1s urgently needed."Seed and soil" hypothesis has been widely acknowledged as the nature law for metastasis during the past decades.The development of cancer metastasis 1s really a complex and multistep process that requires cancer cells to escape from the primary tumor,survive in the circulation while hiding from immune surveillance,seed and grow up at distant sites.However,only a very small portion of primary tumor cells can disseminate to distant organs and subsequent adapt to the microenvironment to form metastasic lesions,which indicating that host environment 1s also important for tumor progression.During the past decades,our research group has been focused on a novel tumor suppressor gene JWA:in the present study,we tried to establish a new strategy to inhibit melanoma metastasis based on the "seed and soil" hypothesis.The JWA gene,also known as ARL6ip5(GenBank AF070523,1998),was initially cloned from primary human tracheal bronchial epithelial cells treated with all-trans retinoic acid(ATRA).JWA is highly conserved during evolution,and is upregulated by environmental stressors such as heat shock and oxidative stress,thus repairing the damaged DNA.The-76G/C variation in the 5'-flanking region of JWA gene is associated with increased odds of leukemias,gastric,esophageal,and bladder cancer risks in Chinese population.And the further functional study revealed that the variation in the-76C allele leads to almost completely the loss of transcription activities stimulated by oxidative stress.JWA inhibits tumor metastasis via several mechanisms,and is regarded as a novel tumor suppressor.It has been demonstrated that JWA activates mitogen activated protein kinase(MAPK)cascade(especially ERK),and differentially regulates cell migrations via downstream effectors including focal adhesion kinase(FAK)and cyclooxygenase-2(COX-2)upon AS2O3 or TPA treatment.JWA modulates transcription factor Sp1 and its downstream effectors(Integrin avp3?ILK and MMP2 signaling)to suppress angiogenesis and metastasis of gastric cancer/melanoma.Based on the study of large independent cohorts of gastric cancer and melanoma patients,we also found that JWA protein levels are significantly downregulated in cancerous compared with adjacent noncancerous tissues.Lower cancerous JWA expression significantly correlates with shorter overall survival(OS),as well as worse clinicopathologic characteristics,such as depth of invasion,lymph node metastasis,TNM stages.In addition,JWA expression level negatively correlates with several other oncogenes(such as FAK,CD31,MMP2,ILK),and has synergistic roles in predicting cancer prognosis.Very recently,a phosphorylation modified JWA peptide(PJP1)screened from JWA amino acid sequence,has been proved to mimic JWA function.PJP1 is injected intratumorally into the nude mice bearing A375(human melanoma cell line)xenografts and inhibits subcutaneous xenograft growth.In addition,the equal amounts of B16F10 melanoma cells are injected into the JWA gene knockout(JWA?/? and wild type(JWA+/+)mice via their footpads or tail vein.Interestingly,host JWA deficiency led to the development of larger tumors in situ and metastatic lymph nodes,as well as more numbers of lung metastatic nodes.Taken together,these results indicate that JWA serves as a tumor suppressor in hindering the "seed" dissemination and creating a harsh "soil" that is not suit for stubborn "seed" blossoms.Objective:To explore the roles of host and tumoral JWA in melanoma metastasis,thus providing the possible clinical application for melanoma treatments.Methods:1.The level-3 mRNA sequencing data(481 samples),level-1 450k DNA methylation data(445 samples),and follow-up records(454 cases)were downloaded from the skin cutaneous melanoma(SKCM)study in The Cancer Genome Atlas(TCGA)(accessed on September 20,2015).The Expectation Maximization(RSEM)normalized JWA expression levels were extracted and transformed before analysis by log2(RSEM+1).Level-1 DNA methylation raw image data were assessed by Illumina Infinium Human Methylation 450 Bead Chips(Illumina Inc.,SanDiego,CA,USA)and were imported into minfi R package for further preprocess and quality control(QC).Unqualified probes were excluded,if they fit the following QC criteria:(?)failed detection(detection P>0.05);(?)unsuccessfully measured in?5%samples;(?)methylated or unmethylated in all samples;(?)common single nucleotide polymorphisms located in probe sequence or in 10-bp flanking regions;or(v)cross-reactive DNA methylation CpG sites(29233 cross-reactive probes).The samples with undetectable probes over 5 percent would be removed as well.After quality control,the 445 samples with 411833 CpG sites were applied for the final analysis.The beta values were further normalized by quantile normalization and followed by beta-mixture quantile(BMIQ)normalization to correct for background,batch effect,and type ?/? probe design bias.The 12 CpG sites were mapped to JWA and were extracted for further analysis.Continuous variables were summarized by mean and standard deviation;the category data were described by n and percentage(%).DNA methylation risk score(MRS)was calculated by the linear combination of the top hits weighted by the corresponding coefficients from the Cox regression.The gene expression and MRS were dichotomized by the optimal cutoff points,respectively,which were discovered using the survival ROC R package to predict 5-year survival.The differences of Kaplan-Meier survival curves were examined by log-rank test and further analyzed by Cox regression with adjustment for age,gender,and stage.The results were described as hazard ratio(HR)per 1%increment of methylation,95%confidence interval(Cl),and P value.2.The PJP1 peptide linked with RGD motif(PJP1-R)was synthesized in order to enhance the target effect 8F labeled PJP1-R(18F-NFP-PJP1-R)were generated and injected intravenously into melanoma xenograft established mice for MicroPET imaging and biodistribution analysis.Pharmacokinetic distribution of PJP1-R was evaluated by intravenous and intraperitoneal injection into the rat.Human melanoma cell line(A375)were subcutaneously injected into nude mice to establish the xenograft models;and murine melanoma cell line(B16F10)were injected into C57BL/6J mice via tail vein to establish the melanoma metastasis model.PJP1-R was injected intraperitoneally into these mice to access its anti-cancer effect.3.Two strains of synthesized plasmids were microinjected into a murine fertilized egg with FVB background JWATet+/-TRE+/-mice were then mated with CMV-Cre mice(JWATet+/-TRE+/-Cre+/-).After intercrossing and/or backcrossing for at least 5 generations,the homozygous transgenic mice with C57BL/6J background(JWATet+/+TRE+/+Cre+/+)were produced.All the genotypes of mice were identified by PCR using the tail DNA as the template.JWATet+/+TRE+/+Cre+/+ mice were fed with 5%glucose with/without 2 mg/mL doxycycline(DOX)every day for one week.In vivo imaging and Western Blot were applied to evaluate JWA expression level.Equal amount of murine melanoma cells were injected into JWATet+/+TRE+/+Cre+/+mice by the tail vein or foot paw.And DOX was applied to increase the expression of JWA in all the organs of mice to evaluate the role of host JWA in melanoma progression.NIH-3T3 cell lines were used to recover the anti-cancer mechanisms of host JWA.4.B16F10 cells were intravenously injected into JWATet+/+TRE+/+Cre+/+ mice to establish the passive metastatic models.Then,these mice were received DOX,PJP1-R or combined treatment.Besides,B16F10 cells were injected into JWA knockout mice via the tail vein,and PJP1-R,p65 inhibitor(Bay 11-7082)and CXCR4 inhibitor(AMD 3100)were used to treated the model mice.In addition,JWA stable knockdown B16F10 cells(As-JWA)and control cells(As-ctrl)were injected into mice foot paw.PJP1-R,p65 inhibitor(Bay 11-7082,20 mg/kg)and CXCR4 inhibitor(AMD 3100,3.5mg/kg)were used to treat the model mice.Results:1.Tumoral JWA transcriptional and DNA methylation level predict overall survival of melanoma patients.1.1 To evaluate the impact of JWA in the prognosis of human melanoma patients,the molecular profiles of JWA from TCGA data portal were systematically explored.The 395 cases,with complete survival follow-up information,DNA methylation,and mRNA sequencing data,were included in the final analysis.All the included cases aged averagely at 58.22�15.52 years,34.18%of them followed to death,63.54%male,52.15%at early stage(Stage ?&?),and 79.75%of tumor tissues collected from metastasis site while only 20.25%from primary tumor site.1.2 The JWA mRNA expression was dichotomized by the 45%percentile which was suggested by survival ROC analysis as 'optimaF cutoff point.The patients with high JWA expression indicated a significant benefit for overall survival(log-rank,P=0.0001),and showed a 45%reduced hazard of death with adjustment for age,gender,and stage(Cox regression,HR=0.55,95%Cl 0.39-0.77,P=0.0005).The subgroup analysis indicated that the significant association was mainly contributed by the metastasis subgroup(Cox regression,HR=0.51,95%Cl 0.36-0.73,P-0.0002).The analysis among the patients at early-stages retained statistical significance(Cox regression,HR=0.47,95%Cl 0.29-0.76,P=0.0022),but showed a moderate result among the late-stages(Cox regression,HR=0.63,95%Cl 0.38-1.05,P=0.0767).1.3 A total of 12 DNA methylation CpG sites mapped to JWA.Two CpG sites were significantly associated with overall survival under the Bonferroni correction for multiple testing(cg20500144,located in an Enhancer near 1st exon,HR=1.42 per 1%increment of methylation,95%CI 1.16-1.73,P=0.001;cg00023132,located 200bp upstream of transcription start site,HR=1.23 per 1%increment of methylation,95%CI 1.07-1.40,P=0.003).The two sites were moderately positively correlated(r2=0.24,P<0.001).To test the independent effect,we re-examined the probe cg20500144 conditioning on cg00023132,which retained statistical significance(HR=1.30 per 1%increment of methylation,95%CI 1.01-1.67,P=0.045).Further,the methylation risk score(MRS)was calculated by the linear combination of the two top hits weighted by their coefficient,respectively,to test the joint effect.The MRS was dichotomized by the 30%percentile�an optimal cutoff from survival ROC analysis.The dichotomized MRS showed a significantly differentiating ability of the patients survival(log-rank test,P=0.0001;HRhigh-vs.low-risk=2.68,95%CI 1.68-4.27,P=0.00003).Similarly,the analysis in the metastasis subgroup attained a significant association(Cox regression,HR=2.51,95%Cl 1.56-4.05,P=0.0001).There were too few samples in the subgroup of primary tumors to illustrate this association.Both the analyses among early-stages(Cox regression,HR=2.77,95%Cl 1.55-4.95,P=0.0006)and late-stages(Cox regression,HR=2.62,95%Cl 1.18-5.81,P=0.0177)got consistent results.1.4 Neither the two single CpG sites nor the joint MRS associated with JWA mRNA expression(P=0.9691).Furthermore,the joint risk score combined of both mRNA expression and MRS showed a higher power to differentiate the patients' outcome(log-rank test,P=1.6 �10-8).The patients in the moderate risk group showed a 2.41 fold increased hazard of death when compared with low risk group(95%CI 1.29-4.51,P=0.0059)and an even higher hazard identified in the high risk group(HR=4.59,95%CI 2.41-8.74,p=3.7�10-6).2.JWA peptide targets melanoma and inhibits melanoma growth and metastasis in vivo.2.1 The PJP1 peptide linked with RGD motif(PJP1-R)was synthesized for further investigation.The observations using micro-PET demonstrated that 18F-labeled PJP1-R(18F-NFP-PJP1-R)specifically assembled in the melanoma xenografts,which suggested that PJP1-R could specifically target melanoma.The biodistribution of 18F-NFP-PJP1-R in nude mice bearing B16F10 xenografts revealed that 18F-NFP-PJP1-R mainly accumulated in the kidney and tumor.2.2 Pharmacokinetic distribution analysis of PJP1-R indicated that Cmax=69998.97 ng/mL,AUC0-t=19519.44 ng/mL�h,t1/2=0.34 h,Vss=369.13 mL/kg,Cl=2556.05 mL/h/kg for intravenous injection in rat;Tmax=0.63 h,Cmax=23065.82�10489.63 ng/mL,AUC0-t=28698.48�285.24 ng/mL�h,t1/2=0.33 h and mean relative bioavailability was 73.35%for intraperitoneal injection in rat.2.3 PJP1-R intraperitoneally injection suppressed melanoma xenografts growth and metastasis in vivo.3.Increased host JWA expression inhibits melanoma growth and metastasis.3.1 The JWATet+/-TRE+/-mice were generated by microinjected the two kinds of Tet on 3G plasmids into a murine fertilized egg with FVB background.Then the JWATet+/-TRE+/-mice were mated with CMV-Cre transgenic mice to knock out the STOP sequence(JWATet+/-TRE+/-Cre+/-).After that,by intercrossing and/or backcrossing the JWA ret+/-TRE+/-Cre+/-mice for at least 5 generations,the homozygous transgenic mice with C57BL/6J background(JWATet+/+TRE+/+Cre+/+)were produced.3.2 JWATet+/+TRE+/+Cre+/+ mice were fed with 5%glucose solution with/without 2 mg/mL DOX every day for one week.In vivo imaging showed that JWATet+/+TRE+/+Cre+/+ mice that were received DOX treatment exhibited GFP fluorescence.Western blot ananlysis confirmed that pulmonary JWA protein level was increased upon DOX treatment.3.3 JWATet+/+TRE+/+Cre+/+ mice were injected with equal amount of B16F10 cells through the footpads.On the next day,JWATet+/+TRE+/+Cre+/+ mice were received DOX treatment to increase JWA expression.The results showed that host JWA inhibited melanoma growth in situ and lymphatic metastasis.3.4 JWATet+/+TRE+/+Cre+/+ mice were received DOX treatment 7 days before,1 day or 7 days after B16F10 cells intravenous injection.The results showed that host JWA significantly inhibited melanoma progression.3.5 JWATet+/+TRE+/+Cre+/+mice were injected with equal amount of B16F10 cells through the tail vein.On the next day,JWATet+/+TRE+/+Cre+/+ mice were received different dose of DOX treatment.The results showed that reduction of pulmonary melanoma metastasis depended on JWA expression.3.6 In the NIH-3T3 cells,JWA promoted IKKp degradation,thus reducing IKK?expression.4.Host and tumoral JWA synergistically inhibits melanoma metastasis.4.1 JWATet+/TRE+/+Cre+/+mice were injected with equal amount of B16F10 cells through the tail vein.Whether DOX,PJPI-R alone or combined treatment could suppress melanoma metastasis to lung,and the combined treatment exhibited better therapeutic efficacy.4.2 B16F10 melanoma cells were injected into eight-week-old JWA?/? mice via the tail vein.PJP1-R(50 mg/kg)was injected(i.p.)daily starting from the next day.p65 inhibitor(BAY11-7082,20 mg/kg)and CXCR4 inhibitor(AMD3100,3.5 mg/kg)were injected 5 times a week(i.p.)starting from the next day.Much more lung metastatic nodules were detected in the JWA?/? mice.Fortunately,PJP1-R,BAY11-7082 and AMD3100 reduced melanoma aggressive metastatic capacity.4.3 Equal amount of JWA stablely knockout down(As-JWA)and control(As-ctrl)melanoma cells were injected into foot paw.PJP1-R(50 mg/kg)was injected(i.p.)daily starting from the next day.p65 inhibitor(BAY11-7082,20 mg/kg)and CXCR4 inhibitor(AMD3100,3.5 mg/kg)were injected 5 times a week(i.p.)starting from the next day.As-JWA melanoma cells developed larger tumor in situ and more metastatic lymph nodes.Interestingly,PJP1-R,BAY11-7082 and AMD3100 reduced melanoma aggressive capacity in vivo.Conclusion:This study is inspired by the "seed and soil" hypothesis.In summary,tumoral and host JWA inhibits melanoma metastasis.In brief,based on the TCGA database,high JWA transcription levels as well as low DNA methylation predicted better prognosis of melanoma patients,which indicating that JWA served as a novel tumor suppressor.Phosphorylated JWA peptide linked with RGD motif(PJP1-R)assembled in melanoma and inhibited melanoma growth and metastasis in vivo.JWA Tet on 3G transgenic mice(JWATet+/+TRE+/+Cre+/+)with C57BL/6J background were generated.DOX treatment increased the JWA expression in all the organs of mice,and inhibited melanoma in vivo by targeting NF-?B-CXCR4 signaling.Increasing tumoral and host JWA expression could synergetically inhibit melanoma metastasis.These findings will provide new targets and strategies for the treatment of melanoma metastasis.