Subsequent studies support the anti-carcinogenesis role of klotho in a variety of human malignancies. The present study investigated the role of klotho on growth and metastasis of osteosarcoma cells. The osteosarcoma cells were transduced with lentivirus particles encoding klotho or scramble control. The reconstructed osteosarcoma cells were injected into the femoral medullary cavity of nude mice to establish a xenograft animal model. The anti-tumor properties of klotho were evaluated in terms of tumor growth, apoptosis, glycogen production, and pulmonary metastasis. Lentivirus-mediated overexpression of klotho significantly decreased tumor volume and weight in osteosarcoma mice. Determination of PCNA and Ki67 expression revealed that overexpression of klotho inhibited cell proliferation in tumor tissues obtained from osteosarcoma xenografts. PAS staining also showed that overexpression of klotho significantly decreased the production of glycogen in osteosarcoma. Moreover, TUNEL positive cells were significantly increased after lentivirus-mediated overexpression of klotho. Furthermore, lentivirus-mediated upregulation of klotho reduced the number of pulmonary metastatic lesions in mice compared to control mice. These findings demonstrated that elevated klotho could inhibit osteosarcoma cell growth and pulmonary metastasis  Osteosarcoma is the most common primary bone malignancies, accounting for 60% of all bone sarcoma. Osteosarcoma has a worldwide incidence of approximately 1∼3 annually per million with 50% of 5-year overall survival. However, the survival of patients with metastatic osteosarcoma has remained only 20% with an overall 5-year survival rate . Cells were maintained in Dulbecco's modified Eagle medium (DMEM)  containing 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 μg/ml penicillin . Cells were cultured at 37 °C in a 5% CO2)/2. Mice were euthanized followed by cervical dislocation at the end point and the tumor samples were measured and collected for subsequent experiments.All animal procedures were approved by the Animal Ethics Committee of our hospital. Six-week-old, SPF-grade nude mice were purchased from Shanghai Slak. Prior to injection, the mice were anesthetized with intraperitoneal injection of pentobarbital. Human osteosarcoma MNNG/HOS cells infected with lentivirus particles encoding klotho  were injected into the femoral medullary cavity of nude mice. In addition, mice in the control group were injected with MNNG/HOS cells and no operative measures were taken on those mice in the blank group. Tumor dimensions  were measured and tumor volumes were then calculated as TV=  according to the manufacturer's protocol. Three independent experiments were conductedExperimental data were analyzed using the GraphPad Prism program . Data are presented as mean ± S.E.M. Statistical significance was performed on the data using ANOVA. A value of P<0.05 was considered to be statistically significant.In order to determine whether klotho was involved in tumor growth, mice were injected with osteosarcoma cells with or without overexpression of klotho, and tumor volume and weight were evaluated. Klotho overexpression in transduced osteosarcoma cells was confirmed by western blot . Four weTo determine whether klotho was associated with glycogen production in tumor cells, mice were injected with osteosarcoma cells with or without overexpression of klotho. Then, PAS staining was used to assess the glycogen content of osteosarcoma with or without overexpressed klotho. We observed no significant changes in glycogen storage between control and blank groups (P>0.05). However, overexpression of klotho significantly decreased the PAS-positive cells per field and thus suppressed the production of glycogen in osteosarcoma .To determine whether up-regulation of klotho was associated with the induction of apoptosis, mice were injected with osteosarcoma cells with or without overexpression of klotho. Then, TUNEL staining was used to assess the apoptotic cell death induced by klotho. Consequently, we detected no significant changes in TUNEL positive cells between control and blank groups (P>0.05). Nevertheless, lentivirus-mediated overexpression of klotho obviously suppressed the TUNEL-positive cell number, suggesting that enhanced expression of klotho was associated with induction of apoptosis in osteosarcoma .in vivo, mice were injected with osteosarcoma cells with or without overexpression of klotho. Subsequently, all injected tumor cells formed large primary tumors at the injection site. Examination of the lungs revealed a high number and volume of metastatic lesions in mice injected with control and blank cells. However, there was a marked reduction in metastases produced by cells overexpressing klotho (in vivo.To clarify the role of klotho in osteosarcoma metastasis g klotho  and B. Tklotho accelerates aging in mice (Klotho is firstly discovered as an anti-aging gene and knock-out of klotho-deficient mice present several bone-associated problems. It has been reported that klotho deficiency in vivo leads to overactivation of Wnt signaling, resulting in stem cell senescence and a complex bone phenotype, which could be prevented by klotho overexpression in this model (Firstly, we investigated whether klotho is associated with tumor growth of osteosarcoma. Previous studies suggest that klotho inhibits many pathways involved in carcinogenesis, including PI3K/Akt/mTOR signaling, Wnt/β-catenin signaling, ERK1/2 signaling, and Smad2/3 signaling (Klotho leads to increased Wnt5A expression, filamin cleavage, and cell migratory potential during melanoma progression (in vivo. Subsequently, examination of the lungs revealed a low number of metastatic lesions in mice injected with lentivirus particles encoding klotho, compared to control and blank cells, supporting the fact that klotho overexpression could suppress the spontaneous pulmonary metastasis of osteosarcoma in vivo.As reported previously, the anti-tumor effects of klotho are attributed to its capacity to suppress migration (in vivo. These findings demonstrate that klotho may serve as a valuable therapeutic target for treatment of osteosarcoma.In conclusion, we have shown that elevated expression of klotho is able to suppress growth and pulmonary metastasis of osteosarcoma


In this study, proteomic profiles of tumor tissues from patients, included anaplastic thyroid carcinoma (ATC) and papillary thyroid carcinoma, and xenografts  were obtained using HPLC-tandem mass spectrometry and compared based on all proteins detected , cancer-related proteins and druggable proteins using pairwise Pearson's correlation analysis. The human tissue showed low proteomic similarity to the ATC cell lines  and to PTC cell lines . Human tissue showed the following similarity to cell lines at the level of 135 cancer-related pathways. The ATC cell lines contained 47.4% of the cancer-related pathways (19.26%-33.33%), while the PTC cell lines contained 40% . In patient tumor tissues, 44-60 of 76 and 52-53 of 93 druggable proteins were identified in ATC and PTC tumors, respectively. Ten and 29 druggable proteins were not identified in any of the ATC and PTC xenografts, respectively. We provide a reference for CDX selecting in While CDX studies have generated valuable insights, CDX tissue and tumor patients differ in their behavior, making CDX inadequate for understanding and predicting human tumor behavior and response to therapy The most commonly used preclinical Selecting appropriate cell lines is essential for accurate cancer studies, but choosing the best cells is often purely empirical. Common cell lines are often used, but their genetic consistency with tumor samples is often unclear. Ovarian cancer, for example, may be better studied using some rarely used cell lines than commonly used lines Towards this goal, various cancer cell lines have been evaluated, as have pooled analysis of various cancers in vivo studies of thyroid cancer.To provide a rational basis for selecting the most appropriate CDX for thyroid cancer studies, we performed proteomics profiling of ATC and PTC tumors as well as several CDX's . The two types of cancer tissue were compared in terms of total proteins, cancer-related pathways and druggable proteins. The results lead us to recommend certain cell lines over others for Fresh frozen samples of primary tumor were obtained from five ATC patients 4 men, 1 woman) and 28 PTC patients  who underwent surgery in Zhejiang Cancer Hospital in Hangzhou, China ; while the PTC cell lines BCPAP and IHH4 were obtained from professor Haixia Guan . Cell lines 8305C and 8505C were cultured in DMEM, while the other lines were cultured in RPMI-1640. In all cases, the medium was supplemented with streptomycin (100 U/mL) and penicillin (100 U/mL) as well as fetal bovine serum (FBS) to a final concentration of 5% (FRO) or 10% . Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO3, animals were sacrificed and tumor tissues were harvested and homogenized. Animal studies were conducted according to the National Research Council's guidelines and approved by the Committee on the Ethics of Animal Experiments of Zhejiang Cancer Hospital and the Center for Drug Safety Evaluation at Zhejiang University.Female BALB/c nude mice weighing 16-22 g [Animal License No. SCXK-(Shanghai)-2015000521727] were obtained from the Shanghai Laboratory Animal Center  and housed at the Center for Drug Safety Evaluation at Zhejiang University . Cultured cell lines were subcutaneously injected into the right flank. Once tumor volumes reached about 100 mmm/z range, 300-2,000; resolving power, 60,000 ; ion spray voltage, 1.8 kV; ion source temperature, 300 ℃; and collision energy, 35 eV. Tandem mass spectrometry was performed with rolling collision energy for the top 20 most intense precursor ions with charge states of at least 2+. Mass and composition of precursor and fragment ions were analyzed using Thermo Xcalibur 3.0 software (Thermo Fisher Scientific).Total proteins from tumor tissues (25 mg) were extracted and digested with trypsin according to the Filter-Aided Sample Preparation method. The digest was desalted and analyzed on a liquid chromatography-mass spectrometry system consisting of a Nano-nLC 1000 system (Thermo Fisher Scientific) connected to a linear quadrupole ion trap-Orbitrap (LTQ Orbitrap Elite) mass spectrometer (Thermo Fisher Scientific), which was equipped with a Proxeon NanoSpray Flex Ion Source (Thermo Fisher Scientific). Peptides were eluted on an Acclaim PepMap RSLC column  using a 150 min-last gradient elution program involving acetonitrile and 0.1% (v/v) formic acid at a flow rate of 250 nL/min. Optimized mass spectrometry conditions were as follows: full scan mass analysis; Piriformospora and Brassica (updated to April 2017). Protein identifications were accepted if the false detection rate (FDR) was <1% and if the score -10×log10 P was >20. The minimum number of unique peptides was set to 1, and the maximum number of post-translational modifications was set to 3. ANOVA was used to assess differences for statistical significance.Raw spectral data were processed and quantitative ratios determined using PEAKS Studio 8 (Bioinformatics Solutions); parent mass tolerance was 15.0 ppm and fragment mass tolerance was 0.8 Da. Results were compared against UNIPROT databases of p < 0.10. The list of 672 human proteins directly related to the mechanisms of action of drugs approved by the US Food and Drug Administration was obtained from Drugbank  not recommended, referring to cell lines showing only weak similarity (r < 0.25) for specific pathways; (2) recommended, referring to cell lines showing strong similarity (r > 0.60), or in the event that all cell lines fail to reach 0.60, the cell line with the highest r-value; (3) priority recommended, referring to the cell line with the highest r-value that is also >0.60.The cell lines were stratified into three classes based on their desirability as A total of 3,961 proteins were identified from tumor samples.After searching 16,167 publications, 4,149 cancer-related genes were identified, of which 2,350 genes were reported by at least two publications. Based on these 2,350 genes, 135 cancer-related pathways were identified . The CDXp < 0.0001) or FRO . BCPAP  seemed slightly more similar to PTC patient tissue than IHH4 .Correlations were also weak based on proteins in cancer-related pathways Figure B. MedianFewer than one of every three pathways showed strong similarity (>0.60) between primary tumors and CDX's; the proportion was 19.26% for 8305C, 33.33% for 8505C, 22.96% for FRO, 25.93% for BCPAP, and 28.89% for IHH4. The two PTC cell lines recapitulated 54 cancer-related pathways (40.0%) with high consistency; the three ATC cell lines, 64 (47.4%). In contrast, the proportion of cancer-related pathways in xenograft tumors showing low consistency (<0.25) with primary tumors was 18.52% in 8305C, 31.11% in 8505C, 22.22% in FRO, 12.59% in BCPAP, and 27.41% in IHH4  and 64 (47.4%) of cancer-related pathways. Overall, these pathways showed moderate similarity between CDX's and human tumors. More than 70% of pathways showed low consistency between the two tumor types. Even in the cell line showing the highest consistency, 8505C, only 33.33% of pathways showed high consistency with primary tumors. Up to one quarter of pathways (11.9-24.4%) showed correlation coefficients <0.25. Priority recommended cell lines were identified for approximately 40 percent of 135 pathways (53 in PTC and 64 in ATC). BCPAP was identified as being not recommended for 15 (11.11%) of pathways with r < 0.25; IHH4, 26 (19.26%) pathways; 8305C, 14 (10.37%) pathways; 8505C for 32 (23.70%) pathways; and FRO, 33 (24.44%) pathways.in vitro as candidates for thyroid cancer treatment The heterogeneous consistency between primary and xenograft tumors at the level of individual pathways suggests that no single CDX model fits most pathways important in thyroid cancer. Our finding that many immunology-related pathways, such as those involving Toll-like receptors, TNF, or NF-κB, show low consistency between primary and xenograft tumor types likely reflects the immunosuppression intrinsic to nude mice. The lack of consistency in the hedgehog pathway between primary and xenograft PTC or ATC tumors is an important reminder of the need to choose cancer cell lines cautiously. The hedgehog pathway is activated to stimulate the growth and spread of many types of solid tumors, including thyroid cancer. It may also affect patient prognosis, but this remains controversial Although few drugs have been approved for treating thyroid cancer, we compared the profile of 672 FDA-approved druggable proteins between primary and xenograft thyroid tumors. These proteins target enzymes, transporters, voltage-gated ion channels, G-protein coupled receptors, nuclear receptors and CD markers. A total of 93 druggable proteins were detected in primary PTC tumors, and only 44% of druggable proteins were expressed in both PTC cell lines. More than 30% of proteins were not detected in either of these lines. A total of 76 druggable proteins were detected in primary ATC tumors, of which 31 were detected in all three ATC cell lines, 22 were detected in at least one line, and 13 were detected in two lines. On the other hand, 10 proteins  were not detected in any of the three lines. The differential expression-level of targeted proteins might partly indicated different response to approved drugs among xenograft models and primary tumors.The proteomic consistency between human thyroid cancer tissue and xenografts derived from human thyroid cancer cell lines was investigated based on total protein, cancer-related proteins, and druggable proteins. The results indicate rather low consistency between primary and xenograft tumors. Most CDX's showed poor correlation with primary tumors (r < 0.4), while the 8505C-derived xenograft showed medium correlation (0.545). At the level of individual cancer-related pathways, fewer than one third of pathways were highly similar between primary and xenograft tumors (>0.60); 12.6-31.1% of pathways showed low similarity (<0.25). Differences in druggable proteins were also observed between primary and xenograft tumors, as well as among xenograft tumors.Key genes related to thyroid carcinoma, such as RET, p53, RAS, BRAF and β-catenin, should be additionally addressed. Therefore, the responding signaling pathway, included p53 signaling pathway, RAS signaling pathway, MAPK signaling pathways, WNT signaling pathway, and PI3K-AKT/RAS signaling pathway were additionally discussed. Most signaling pathways were with moderate correlation (r=0.40-0.53) between xenografts and patients, except poor consistancy of p53 in PTC (r=0.23).There're also limitations of subcutaneous xenograft tumor model. For example, subcutaneous tumor size variability was generally very low, and the proliferation rate was slower than orthotopic tumor model. These differences in overall growth indicated the crucial role of an appropriate microenvironment and organ-specific angiogenesis in facilitating initial tumor growth and perhaps later aggressiveness. Another important reason is that subcutaneous xenograft tumor model do not clearly reproduce the primary site of cancers and nor do they represent the common sites of metastasis. On the contrary, orthotopic tumor placement appears to better mimic the microenvironment, morphology, growth, and metastatic patterns of human cancer. Therefore, these limitations may also be important reasons for the poor correlation between the CDX model and the primary model in thyroid tumor in our study. Moreover, the mutation status, express levels of key genes should be focus in future studies.Supplementary figures and tables.Click here for additional data file.


Insights into the diversity and relationships among elite breeding materials are an important component in maize improvement programs. We genotyped 63 inbred lines bred for high levels of provitamin A using 137 single nucleotide polymorphism markers. A total of 272 alleles were detected with gene diversity of 0.36. Average genetic distance was 0.36 with 56% of the pairs of lines having between 0.30 and 0.40. Eighty-six percent of the pairs of lines showed relative kinship values <0.50, which indicated that the majority of these provitamin A inbred lines were unique. Relationship pattern and population structure analysis revealed presence of seven major groups with good agreement with Neighbour Joining clustering and somewhat correlated with pedigree and breeding origin. Utilization of this set of provitamin A lines in a new biofortification program will be aided by information from both molecular-based grouping and pedigree analysis. The results should guide breeders in selecting parents for hybrid formation and testing as a short-term objective, and parents with diverse alleles for new breeding starts as a long-term objective in a provitamin A breeding program. The leaf samples were sent to LGC Genomics in the UK for DNA extraction using LGC’s beadex™ extraction chemistry and genotyping using KASP platform. The 63 samples were genotyped with 142 SNPs and the results were visualized through the SNPviewer software (https://www.lgcgroup.com/products/genotyping-software/snpviewer). The SNPs selected for this study were recommended for routine low-cost genotyping . These lines were selected based on high provitamin A content and combining ability. The procedure for developing these inbred lines was described in detail by Pixley et al. . A singlGene diversity, polymorphic information content (PIC), allele frequency, and relative kinship were computed using the TASSEL software package . Gene diversity in this set of lines ranged from 0 to 0.50 with an average of 0.36 while heterogeneity ranged from 0 to 0.18 with an average of 0.05. The PIC value ranged from 0 to 0.37, with an average of 0.29. Genetic distance between pairs of inbred lines ranged from 0 to 0.54, with an average of 0.36. Most (34.2%) pairs of lines in the present study had genetic distances between 0.35 and 0.40 . Relative kinship coefficients between pairs of lines ranged from 0 to 0.66, with an average of 0.42 , but most values (86%) fell between 0.35 and 0.50.The neighbor-joining (NJ) tree generated from Rogers GD matrix was constructed to gain more insight into the genetic diversity among the set of maize lines. The dendrogram grouped the 63 lines into three major groups with seven subgroups . The firThe results from K = 7 were compared with pedigree and breeding history to characterize the clusters, and it was seen that the STRUCTURE and NJ cluster classifications in many cases placed lines descended from a common ancestor in several groups. For example, lines derived from CML537 were in groups 2 and 4. Two lines in group 4 (entries 46 (CLHP0235) and 47 (CLHP0221)) involved a backcross to CML537 and the donor for carotenoid concentration, while the two lines in group 3 (entries 48 (CLHP0213) and 56 (CLHP0003)) differed from those in group 2 based on the donor for carotenoid concentration. Therefore, the distribution of these lines across the two groups could be attributed to the breeding history in carotenoid conversion program. A similar scenario was applicable to derivatives from line CLQRCWQ109  that were spread across groups 2, 3 and 5, and for the majority of lines from CML506  (Table S1). The exceptions were lines derived from CML496 and CML486 that were placed in two separate groups despite the fact that the same donor line for carotenoid concentration was used in each case. Thus, clusters must be assumed to form based on the combined contribution of many parents, and are not formed based on any one ancestral line or phenotypic trait (such as carotenoid concentration). The clusters formed by both NJ dendrogram and STRUCTURE analysis were fairly clearly delineated, but a PCA of these data  did not show clear separations at all .Utilization of diverse sources of provitamin A maize lines with different carotenoid content is important for a new biofortified maize genetic enhancement program. Use of molecular markers to infer genetic diversity in yellow endorsperm and provitamin A maize has been reported in many studies (Menkir et al. The relative kinship coefficient reflects the approximate degree of identity between two given individuals (Dao et al. In conclusion, there was moderate genetic distance among some of the provitamin A lines used in this study, more than expected due to the common parents used as donor lines for carotenoid content. The results of this study indicated that a combination of molecular marker analyses, pedigree/breeding history, and directed testcrossing is needed to assign inbred lines to sub-groups for breeding decisions. There is substantial genetic variation in this set of provitamin A lines which will be useful for a new maize biofortication program in Uganda.


In addition, ribociclib induces apoptosis of RCC cells, but with less potency compared to its antiproliferative activity. The combination of ribociclib with chemotherapeutic or immunotherapeutic agents is synergistic in RCC cell lines. Of note, ribociclib demonstrates selective anti-RCC activity by sparing normal kidney cells and fibroblast cells. Consistent with the in vitro findings, ribociclib inhibits RCC growth at the dosage that does not lead to toxicity in mice and enhances the in vivo efficacy of RCC standard-of-care drugs. Mechanistically, we show that ribociclib remarkably inhibits phosphorylation of retinoblastoma protein (Rb) at various sites, leading to the suppression of transcription of E2F target genes in RCC cells. Our findings clearly demonstrate the potency and selectivity of ribociclib in RCC preclinical models, via inhibition of the CDK4/6-cyclin D/Rb pathway. Our findings support a clinical trial for the combination of ribociclib with chemo/immunotherapy in RCC.Renal cell carcinoma (RCC) is the most aggressive type of genitourinary cancer and is resistant to current therapies. Identifying drugs that enhance the efficacy of RCC standard-of-care drugs at sublethal concentrations is an alternative therapeutic strategy. Ribociclib is an orally available cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor that is approved for the treatment of breast cancer. In this work, we demonstrate that ribociclib at clinically achievable concentrations inhibits proliferation of 7 out of 9 tested RCC cell lines, with IC RCC is e in RCC . IdentifThe retinoblastoma (RB) tumor suppressor is a known master regulator of the cell cycle that slows cell division by preventing the progression of the cell cycle from the G1 to S phase . The phoin vitro and in vivo efficacy of ribociclib alone and its combination with RCC standard-of-care drugs. In addition, we attempted to identify the mechanism of action of ribociclib in RCC cells focusing on Rb signaling.In this work, we investigated the 2 and 20% O2. Interferon-α  was reconstituted in water. Ribociclib  and 5-Fluorouracil  were reconstituted in DMSO. For single drug treatment, ribociclib at concentrations ranging from 0.05 to 0.8 μM was added to the well. For combination studies, ribociclib, 5-FU, and IFN-α alone at one single dose, the combination of ribociclib with 5-FU, and the combination of ribociclib with IFN-α were added to the well.Seven human RCC cell lines , three human immortalized normal kidney cell lines , and a normal human fibroblast cell line (BJ) were obtained from ATCC. Two human RCC cell lines SW839 and UM-RC-2 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cell lines were maintained in the Key Laboratory of Hubei University of Arts and Science. Cells were cultured in Eagle's Minimal Essential Media (MEM) supplemented with 10% fetal bovine serum , 1% HEPES , and penicillin/streptomycin in a 37°C atmosphere with 5% CO3 cells/well were seeded to a 96-well plate. The next day, drugs were added to the well and incubated for 72 hours. Cell proliferation activity was assessed using the Bromodeoxyuridine (BrdU) Cell Proliferation Assay Kit as per the manufacturer's protocol.5 × 105 cells/well in a 12-well plate were seeded. The next day, drugs were added to the well and incubated for 72 hours. The treated cells were trypsinized and resuspended in PBS. Cells were stained using the Annexin V-FITC/7-AAD  Kit as per the manufacturer's protocol. The stained cells were analysed on Beckman Coulter FC500 with a minimum of 10,000 events counted. Annexin V+/7-AAD- and Annexin V+/7-AAD+ cells were considered apoptotic cells.5 × 106 cells/well in a 6-well plate were seeded. The next day, drugs were added to the well and incubated for 24 hours. The treated cells were lysed at 4°C in radioimmunoprecipitation assay (RIPA) buffer . Insoluble materials were cleared by centrifugation, and the supernatant was collected for protein concentration measurement using a BCA protein assay kit . An equal amount of proteins was resolved by SDS-PAGE and was transferred to a PVDF membrane. Total Rb, phosphor Rb, and p16INK4a were detected using antibodies purchased from Santa Cruz Biotechnology, Inc.5 × 106 cells/well in a 6-well plate were seeded. The next day, drugs were added to the well and incubated for 24 hours. Total RNA in the treated cells was isolated using TRIzol . RT-PCR was performed using the Superscript One-Step RT-PCR kit  as per the manufacturer's protocol. Primer sequences are as follows: FOXM1—forward: 5′-GGT GTG AAT GAA GAC TTG GCT GA-3′ and reverse: 5′-GTT TCA TCC AGG ATG GCT TGG CA-3′, CCNE1—forward: 5′-ACG AAG GTC TGC GCG TGT T-3′ and reverse: 5′-CCG CTG GCC ATG AAC TAC CT-3′, and CDC6—forward: 5′-TGT CAA AAG CCA GAC TAT-3′ and reverse: 5′-GTG AAT AAG ACC AAC CCT-3′.5 × 103), mice were randomized into the following six groups (n = 10 per group): (i) control arm receiving 20%/80% DMSO/saline, (ii) intraperitoneal 5-FU at 1 mg/kg once every three days, (iii) intraperitoneal 105 units of pegylated IFN-α  once every 3 days, (iv) oral ribociclib at 50 mg/kg daily, (v) combination of ribociclib and 5-FU, and (vi) combination of ribociclib with IFN-α. The dosing interval was determined according to published literatures as well as our preliminary results. The dose of a drug that leads to slight or moderate inhibition of tumor growth (<40%) as a single drug alone was chosen for combination studies. All mice were euthanized at the end of the 3-week treatment.The animal experiments conformed to the guidelines set forth by the Institutional Animal Care and Use Committee of Xiangyang Central Hospital. RCC xenografts were generated by subcutaneous injection of 1 million 786-O or CaKi-1 cells into the flank of 6-week-old male NOD/SCID mice. Tumor size and body weight were monitored every alternative day. After the development of palpable tumors  for a panel of RCC and normal cell lines were evaluated using a range of concentrations from 0.0125 to 1.6 μM. All the concentrations tested in our study are clinically achievable as the plasma concentration of ribociclib up to 6 μg/ml was detected in patients receiving ribociclib therapy [50 range of ribociclib in these 7 RCC cell lines was from 76 to 280 nM  and fibroblast cells (BJ)  tested (μM induced 19% to 56% apoptosis in 7 out of 9 RCC cell lines (The inhibitory effects of ribociclib and its 50% inhibitory concentration (IC therapy . Nine hu therapy . Ribociclls (BJ) . The IC5) tested . We furtll lines . Based oNCT03078751 and NCT03822468), we determined the combinatory effects of ribociclib with RCC standard-of-care drugs: the immunotherapeutic agent IFN-α and the chemotherapeutic agent 5-FU [α and 5-FU in those RCC cell lines that are sensitive to ribociclib as a single drug alone. In addition, ribociclib did not enhance the efficacy of IFN-α and 5-FU in those RCC cell lines that are resistant to ribociclib. These results suggest that the combination achieves greater efficacy than the single drug alone only in RCC cell lines that ribociclib is active against.To correlate with clinical trials that test the efficacy of ribociclib in the addition of other anticancer agents , ovarian cancer (NCT03056833), and advanced neuroendocrine tumors (NCT03070301). Our findings may accelerate the initialization of clinical trials on the safety and efficacy of ribociclib and standard-of-care drugs in patients with RCC.We show that ribociclib is a potential candidate for RCC treatment because ribociclib at clinically achievable concentration (1) potently inhibits growth in multiple RCC cell lines , 2) dis dis2 andThe principal function of cyclin D-CDK4/6 activity is to initiate the phosphorylation of Rb at sites like Ser807/811 and Ser795 that provoke functional inactivation of Rb and the subsequent release of E2F transcription factors which drive the expression of genes that support DNA synthesis and S phase progression , 31. ConIn conclusion, our work is the first to demonstrate the selectivity and synergistic effects of ribociclib with RCC standard-of-care drugs in preclinical settings. Ribociclib has advantages over many other investigational compounds given the well-known information on drug molecular target, toxicity, and pharmacokinetics. Our findings support further investigation of ribociclib in RCC in clinical settings.


Therapeutic options for the treatment of men with metastatic castration-resistant prostate cancer are limited. Docetaxel—a taxane-based chemotherapeutic agent—was the first treatment to demonstrate significant efficacy in the treatment of this disease. However, responses to docetaxel are frequently curtailed by development of drug resistance, and patients eventually succumb to disease progression due to acquisition of drug resistance. In this study, we established drug-resistant prostate cancer cell lines and identified several mechanisms that may be associated with the development of drug resistance in prostate cancer. Actioning these mechanisms could provide a potential approach to re-sensitize drug-resistant cancer cells to docetaxel treatment and thereby further add to the life-prolonging effects of this drug in men with metastatic castration-resistant prostate cancer.Docetaxel—a taxane-based chemotherapeutic agent—was the first treatment to demonstrate significant improvements in overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, the response to docetaxel is generally short-lived, and relapse eventually occurs due to the development of resistance. To explore the mechanisms of acquired docetaxel resistance in prostate cancer (PCa) and set these in the context of androgen deprivation therapy, we established docetaxel-resistant PCa cell lines, derived from the androgen-dependent LNCaP cell line, and from the LNCaP lineage-derived androgen-independent C4-2B sub-line. We generated two docetaxel-resistant LNCaPR and C4-2BR sub-lines, with IC50 values 77- and 50-fold higher than those of the LNCaP and C4-2B parental cells, respectively. We performed gene expression analysis of the matched sub-lines and found several alterations that may confer docetaxel resistance. In addition to increased expression of ABCB1, an ATP-binding cassette (ABC) transporter, and a well-known gene associated with development of docetaxel resistance, we identified genes associated with androgen signaling, cell survival, and overexpression of ncRNAs. In conclusion, we identified multiple mechanisms that may be associated with the development of taxane drug resistance in PCa. Actioning these mechanisms could provide a potential approach to re-sensitization of docetaxel-resistant PCa cells to docetaxel treatment and thereby further add to the life-prolonging effects of this drug in men with mCRPC. Prostate cancer (PCa) is the most common cancer among men in developed countries and a major cause of male mortality, mostly due to disease progression to metastatic castration-resistant prostate cancer (mCRPC) ,2. AlthoR and C4-2BR, respectively) and characterized them in terms of proliferation, viability, gene dosage, cell cycle, and resistance to docetaxel. We also performed genome-wide gene expression profiling of the two parental PCa cell lines (LNCaP and C4-2B) and their docetaxel-resistant derivatives and identified a number of mechanisms that may be associated with the development of taxane drug resistance.To identify mechanisms of resistance that could lead to the discovery of biomarkers able to predict docetaxel resistance, or of novel therapeutic targets, we developed and characterized docetaxel-resistant PCa cell models. Given that docetaxel is primarily relevant for treatment of PCa in the metastatic setting, we used the LNCaP and C4-2B PCa cell lines, as these lines constitute a cellular model of PCa progression that mimics the natural history of the disease. LNCaP is a poorly tumorigenic, androgen-sensitive, and non-metastatic PCa cell line, derived from a needle biopsy taken from the left supraclavicular lymph node of a 50-year-old Caucasian male patient . A castrLNCaP and C4-2B PCa cells, kindly provided by Prof. Flores-Morales , were cultured and maintained in RPMI-1640 medium + glutaMAXTM-I  supplemented with 10% fetal bovine serum (FBS). LNCaP and C4-2B docetaxel-resistant cell lines were generated by exposing them to an initial dose of 2.5 nM docetaxel (suspended in dimethyl sulfoxide (DMSO)) for 48–72 h. After each treatment, cells were allowed to fully recover before assessing their resistance to docetaxel and any experimental work. LNCaP cells were exposed to docetaxel through 38 passages over 320 days at concentrations ranging from 2.5–70 nM. C4-2B cells were exposed to docetaxel through 45 passages over 315 days at concentrations ranging from 2.5–100 nM. Untreated parental cells were cultured alongside as an appropriate control to ensure the resistant phenotype alterations. Batches of cells in different passages and statuses of docetaxel resistance were frozen down after assessing their resistance. All cell lines were tested for mycoplasma contamination using Mycoplasma PCR detection kit  and no infections were observed.https://david.ncifcrf.gov/), a web-based statistical hypergeometric test applied for enrichment analysis of Gene Ontology (GO) categories. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.RNA expression was assessed using human Affymetrix Clariom D arrays . Arrays were analyzed using the SST-RMA algorithm in the Affymetrix Expression Console Software. Differentially expressed genes were identified using significance analysis of microarrays (SAM) to control the false positive error rate . All genCytotoxicity was performed using the 3--2,5-diphenyl-tetrazolium bromide (MTT) assay as previously described . BrieflyCell adherence and proliferation were monitored in real time using the xCELLigence system real-time cell analysis (RTCA) E-Plate . The xCELLigence RTCA DP instrument was used according to the manufacturer’s instructions. Briefly, after equilibration to 37 °C, E-plates were placed into the xCELLigence RTCA DP instrument, and the baseline impedance was determined to ensure that all connections were working within acceptable limits across the plate. For each experiment, 10,000 LNCaP and 8000 C4-2B cells were seeded in each well of an E-plate. The impedance value of each well was automatically monitored by the xCELLigence system for a duration of 140 h and is presented as a normalized cell index value.Cells were plated for 24 h in 6-well plates at a density of 200,000 cells/well. After plating for 24 h, serum starved in RPMI-1640 medium + glutaMAXTM-I  was added for an additional 24 h to synchronize the cells. Cells were treated for 24 h, followed by wash with PBS and trypsinization. The single cell suspension was collected and centrifuged at 1200 rpm for 5 min at 5 °C, and pellets were resuspended and fixed with 96% cold ethanol. Samples were centrifuged and resuspended in 100 μL staining buffer containing 0.25% Triton (Sigma-Aldrich) in PBS, 25 μg/mg RNase A , and 10 μg/mL PI (Sigma-Aldrich) and incubated for 30 min at 37 °C. Cell cycle progression was measured by BD FACSVerseTM flow cytometer . Data were analyzed in Flowjo 10.1 Single cell Analysis Software .g for 10 min at 4 °C and supernatants were collected. Total amount of protein was assessed by the PierceTM BCA Protein Assay Kit (Thermo Scientific), according to the manufacturer’s instructions. The Novex® NuPAGE® MES SDS Running Buffer (Thermo Scientific) was used for separation of proteins according to manufacturer’s instructions. Samples were loaded onto precast 10- or 15-well 4–12% Bis-Tris Gel gels . Proteins were blotted onto a nitrocellulose membrane  using an iBlot®2 gel transfer device. Blots were blocked for 1 h in washing buffer (PBS + 0.1% Tween 20) containing 5% non-fat dry milk and incubated overnight with the appropriate primary antibody diluted in blocking reagent: Anti-PgP ; Anti-TRAIL antibody (MAB687) ; Anti-β-Actin Antibody  (BD Bioscience); anti-cleaved Caspase-3 ; Purified Mouse Anti- P150 . After washing 3 × 10 min in TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Mouse/Rabbit) for 1 h at RT. Membranes were washed 3 × 10 min in TBS-T and developed using Clarity Western ECL substrate (Bio-Rad) detection reagent. Protein bands were detected with the UVP BioSpectrum Imaging System . The Peggy SueTM system  was used to detect and quantify Anti-PgP  and Anti- AR Antibody (441): sc-7305 , both diluted 1:50. Samples were prepared and loaded in a 384-well assay plate according to the manufacturer’s instructions, and data were analyzed using Compass software Compass for SW softwareVersion 5.0.1, Protein Simple, Oxford, UK).Whole cells were harvested and lysed using lysate buffer M-PER Mammalian Protein Extraction Reagent  supplemented with Pierce Protease and Phosphatase Inhibitor Mini tables (Thermo Scientific). Cell lysates were centrifuged at 14,000× 2O, before being incubated for 1 h with primary ABCB1 antibody (Abcam) diluted 1:1000 in antibody diluent with background reducing components (Dako). Then, sections were washed twice in TBS + 0.5% Triton X-100 and incubated for 20 min with a High Definition Polymer Detector . Colorimetric signals were detected using DAB. Sections were developed with EnvisionTM FLEX DAB + Chromogen (Dako) diluted in EnvisionTM FLEX Substrate Buffer to visualize the primary antibody. Sections were counterstained with Mayer’s hematoxylin  and mounted with Pertex xylene-based mounting media (Hounisen).For the immunocytochemistry, cells were fixed in 10% PBS buffered formalin (VWR) and left to incubate at 4 °C overnight before being cut into 3 μm sections and placed on glass slides. After being deparaffinized and rehydrated, sections were exposed to induced antigen retrieval to unmask epitopes. Sections were boiled for 10 min in Envision Flex Target Retrieval Solution, high pH , diluted 1:50 in miliQ HParental and docetaxel-resistant cells lines (at similar passage) were harvested and the chromosomes were banded as described previously . NomenclABCB1 gene copy was assessed by FISH using LNCaP and C4-2B, both parental and resistant cell lines. Cells and slides were prepared as previously described [ABCB1 gene and centromere of chromosome 7, according to the manufacturer’s instructions. A fluorescence microscope  with a Texas Red/FITC double filter was used for quantifying red (ABCB1) and green (chromosome 7) signals.escribed . FISH waR and LNCaPR, respectively) by continuous exposure (over a period of 10 months) of C4-2B and LNCaP cells to sub-lethal, stepwise increasing concentrations of docetaxel (from 0.1 to 100 nM). Cell viability assays established docetaxel IC50 values of 99.47–100.50 and 49.50–50.65 nmol/L for C4-2BR and LNCaPR, respectively, compared to the C4-2B and LNCaP parental cells that had IC50 values of 1.00–1.40 and 0.78–1.06 nmol/L, respectively -stained cells exposed, or not, to 20 nM docetaxel. Exposure to docetaxel for 24 h induced G2/M arrest in both the C4-2B and LNCaP parental cells, while no significant effect was observed on the resistant cells . As shown in R line, growth rates were slightly higher than those of the parental cell line  of three independent passages of C4-2BR and LNCaPR and the matched parental C4-2B and LNCaP cells, respectively. Cells were grown in the absence of docetaxel to identify stably acquired changes.To identify those genes that may contribute to docetaxel resistance in C4-2BR cells compared to the matched parental C4-2B cell line, whereas only 152 genes were significantly deregulated in LNCaPR compared to the matched parental LNCaP cell line. A complete list of deregulated genes in both cell lines is provided as We found a total of 1300 significantly deregulated genes in C4-2BR resistant cells compared to the matched parental C4-2B cell line was performed  in our docetaxel-resistant sub-lines. We found cell line-specific changes in the profile of the AR-regulated genes, concomitant with acquisition of docetaxel resistance. In the androgen-independent C4-2BR line, AR-regulated genes were significantly down-regulated compared to the matched parental cell line , whereas we found no differential expression of AR in docetaxel-resistant LNCaPR cells compared to the matched parental cell line .Since alterations in androgen receptor (AR) signaling are known to be critical in PCa progression, and resistance to docetaxel has been associated with deregulated AR signaling ,15, we sell line a. By con(R cells a. FurtheAR mRNA and AR protein levels previously observed in LNCaP cells [R cells (1.7-fold) while it increased slightly in the LNCaPR cells (1.2-fold) compared to their respective matched parental cell lines . The LNCaPR docetaxel-resistant cells, on the other hand, which up-regulated AR signaling, did not differ significantly from the parental cells or, if anything, were only slightly more sensitive to enzalutamide than the matched parental cells  gene, was found to be up-regulated at the mRNA level in both the C4-2BR and LNCaPR docetaxel-resistant cell lines. ABCB1/MDR1 is an ATP-dependent efflux pump that decreases the intracellular concentration of a variety of anti-cancer drugs, leading to multidrug resistance in several types of cancer, including PCa [R and LNCaPR docetaxel-resistant cell lines (R in R +RHD123). To establish that these differences in RHD123 accumulation were due to efflux activity by ABCB1, resistant cells were exposed to PSC833, a cyclosporin A analog and ABCB1 inhibitor [R +RHD123 +PSC833).To address common and, presumably, more prevalent mechanisms of resistance, we examined the 30 genes we found to be deregulated in both cell lines . ABCB1, ding PCa ,26. Westll lines a. Overexll lines b. We thenes R in c. Due tones R in c, C4-2B.nhibitor , prior tR and LNCaPR cells, we measured cell viability after docetaxel treatment for 48 h in parental and resistant cells while concomitantly blocking ABCB1 with increasing concentrations of PSC833 , no major differences were seen on ABCB1 expression, indicating that the final levels of resistance to docetaxel were a result of a compounded effect of multiple mechanisms, one of which was ABCB1 up-regulation. It was conceivable that this abrupt increase in ABCB1 expression was due to chromosome 7 abnormalities [ABCB1 in our model system, karyotypical analyses of metaphase chromosomes, stained by Giemsa for G-bands, were performed on parental as well as resistant cell lines. However, as shown in ABCB1 gene and therefore overexpression of ABCB1. Both C4-2BR and LNCaPR cells maintained the parental chromosome 7 ploidy, despite an up-regulation of ABCB1 mRNA expression of 20-fold and 10-fold, respectively.Although reduced efficacy of taxanes has been previously associated with ABCB1 expression, the specific mechanisms responsible for increased expression of ABCB1 in acquired drug resistance are not completely clear. Different mechanisms of 1 region . Abnormaof ABCB1 ,30,31,32malities . To deteABCB1, no gene copy number gain difference was observed  paralogues, vtRNA1-1 and vtRNA1-2, as being strongly up-regulated in the C4-2Bpathways  and convpathways . Thus, oTNFSF10/TRAIL) gene was significantly up-regulated in LNCaPR cells compared to its parental counterpart , albeit to a much lower extent. However, Western blot analysis revealed TRAIL protein overexpression not only in LNCaPR cells but also in C4-2BR cells compared to their matched parental counterparts (We further scrutinized our data for genes related to cell death pathways and found that the Tumor Necrosis Factor superfamily member 10 (nterpart . Expressterparts a.We then examined whether expression of TRAIL was affected by ABCB1 efflux function in our docetaxel-resistant cell lines. Blocking ABCB1 activity with PSC833 (1 μM) for 48 h induced TRAIL protein expression further in both docetaxel-resistant cell lines a, which R cells , we found that C4-2BR or LNCaPR cells.To further evaluate whether ABCB1 efflux activity, rather than expression, was a determinant of sTRAIL sensitivity, we measured the cytotoxic effect of sTRAIL on parental and resistant cells while blocking ABCB1 efflux activity c, using R and LNCaPR, together with their respective parental lines, C4-2B and LNCaP, respectively, constitute useful cellular models to study docetaxel resistance in PCa in the context of disease progression from localized, hormone-dependent PCa to mCRPC. Gene expression profiles of these resistant models identified likely mechanisms involved in acquisition of resistance to docetaxel (We generated a set of docetaxel-resistant PCa cell lines (LNCaPR and C4-2BR) based on a cellular model of PCa progression . The resocetaxel . We discocetaxel , up-reguocetaxel , and derocetaxel .Our data showing that resistance to docetaxel was associated with AR signaling are consistent with results from the CHAARTED  and STAMPEDE  clinical trials, which suggested an association between androgen receptor signaling and docetaxel sensitivity in PCa ,41. SecoR cells, compared to LNCaPR cells. Given that the LNCaP cell line represents the androgen-sensitive and non-metastatic setting of PCa, whereas C4-2B embodies the metastatic and androgen-independent setting of the disease, inference from our model would suggest that development of resistance to docetaxel may lead to enhanced or suppressed AR signaling, depending on the setting of the disease, which has implications for clinical decision making. As novel treatments become available, physicians need to decide how best to sequence available treatments. Currently, there are no conclusive clinical data providing direct evidence on how best to sequence post-docetaxel treatments in CRPC. If our model system holds true, patients with CRPC who received docetaxel, when developing resistance, will become less responsive to hormonal treatments, such as enzalutamide. Indeed, enzalutamide showed a better effect in the post-docetaxel setting in our model, when docetaxel was used in the androgen-sensitive setting , a RAS family protein involved in the MAPK cascade pathway [RUNDC3B gene is nested in the same genetic locus as ABCB1, with several RUNDC3B exons located in the complementary strand of the ABCB1 gene, and expression of RUNDC3B is associated with alternative regulation of the ABCB1 promoter and expression of ABCB1 mRNA isoforms [ABCB1 and RUNDC3B genes, such as an epigenetic event. As both cell lines displayed a similar behavior, with concomitant overexpression of ABCB1 and RUNDC3B genes, it is conceivable that this mechanism may be a common one, and if it is actionable, it may provide an attractive target for preventing development of drug resistance.We also found that overexpression of the ABCB1 efflux pump was strongly associated with development of docetaxel resistance in both cell lines in our model system . Overexpsistance ,31,32,33ll lines , indicat pathway . The RUNisoforms ,46,47. AR docetaxel-resistant cells as compared to their parental counterparts, with log2 ratios of approximately 139 and 20, respectively. Vault RNAs can recognize and bind chemotherapeutic compounds, thus preventing them from accessing their target sites. As a result, vtRNAs have been associated with chemoresistance in cancer cells [TNFSF10/TRAIL, which we found up-regulated in our drug-resistant cell lines  paralogues, vtRNA1-1 and vtRNA1-2, as being strongly up-regulated in the C4-2Ber cells ,48,49,50er cells ,52. In aer cells  and convll lines . Althougll lines a. Taken R cells and 152 deregulated genes in LNCaPR cells compared to the corresponding parental lines. Here, we focused primarily on a few of those genes that showed a common pattern of deregulation between the two cell lines (CDKN1A), or p21Cip1, a cell cycle regulator and tumor suppressor, overexpressed in both LNCaPR and C4-2BR. Increased expression of CDKN1A should induce cell cycle arrest and reduce proliferative activity. However, we found that LNCaPR cells actually showed slightly higher proliferation rates than the parental cell line, whereas C4-2BR cells had a lower proliferation rate than the corresponding parental cell line (We found a total of 1300 deregulated genes in C4-2Bll lines , and forell line d, suggesWe generated and characterized two docetaxel-resistant prostate cancer sub-lines. We, like many others previously, identified ABCB1 gene up-regulation as a common mechanism involved in acquired docetaxel resistance. However, we could also identify multiple additional mechanisms, associated with AR signaling and regulation of cell death processes, which may contribute to docetaxel resistance and could be necessary to complement up-regulation of ABCB1 to achieve resistance to high concentrations of docetaxel.Comparison of our data with similar datasets generated from other docetaxel-resistant derivatives of C4-2B and LNCaP cells generated by other researchers, as well as from other PCa cell lines, should provide further knowledge as to whether acquisition of resistance is achieved by deregulation of a finite number of specific genes, or if one, rather, should enumerate all the traits that have been found to be associated with resistance under an overarching organizing principle, or hallmarks of resistance.


CRBN E3 ligase complex [Immunomodulatory compounds, IMiDs® lenalidomide and pomalidomide mediate their anti-myeloma activities via cereblon, a component of the Cul4A complex , 2. HereWe observed order of antiproliferative activity of iberdomide > pomalidomide > lenalidomide in matched lenalidomide-sensitive (H929) and an acquired lenalidomide-resistant (H929/LR) cell line Fig. S. In a paPomalidomide and lenalidomide bind cereblon with similar affinity (~3 µM) . We prevCurrent clinical application of IMiDs compounds include doublet and triplet combinations with dexamethasone, bortezomib, and/or daratumumab. We initially compared the antiproliferative and pro-apoptotic activity of iberdomide to pomalidomide in combination with bortezomib in MM1.S cells. Due to the potent cytotoxic effects of bortezomib, pomalidomide, and iberdomide, and the narrow window of observable combinatorial effects, we titrated either pomalidomide (0.001–10 μM) or iberdomide (0.0001–1 μM) against bortezomib (0.0625–1 nM) Figs. S, left. UWhile the combination of iberdomide with bortezomib displayed strong antitumor effects, a potential clinical combination would likely include dexamethasone. Proliferative inhibition in MM1.S cells with the combination of iberdomide/bortezomib Fig. S, left, fIn presence of human-derived complement, iberdomide plus daratumumab had a greater inhibitory effect on H929 cells than either drug alone Fig. S. While cn = 9) using methods previously described [CRBN gene mutation status in the cell lines by NGS. Interestingly, in three cell lines there were alterations in the CRBN gene (Table CRBN transcript with a subsequent deletion of exon 6 of CRBN (CRBN∆6). The CRBN∆6 protein product was detectable by western running at a slightly smaller molecular weight  cell lines n =  using meescribed  cells . In seven of the ten PR cell lines where cereblon expression was reduced but detectable   (data not shown).Next, we tested iberdomide activity in PR cell lines with respect to cereblon levels and mutations. To do that, relative cereblon protein expression levels in the PR lines were compared to the isogenic sensitive parental lines as determined by western and densitometry (100% representing no change)  Fig. S, there wTo gain insight into the potential mechanisms of iberdomide in PR cells, we analyzed Aiolos degradation in KMS12BM and KMS12BM/PR lines treated with either pomalidomide or iberdomide. As expected, both pomalidomide and iberdomide led to rapid Aiolos depletion in the KMS12BM line Fig. . In contTo evaluate the immunomodulatory effects of iberdomide in PR cells, we performed PBMC co-culture killing experiments. Iberdomide equally induced PBMC-mediated killing of both parental MM1.S cells and MM1.S/PR cells Fig. . This isn = 10; 9 of 10 were refractory to pomalidomide)  showed significant decreases in substrate proteins during iberdomide therapy  is ongoing to determine the maximum tolerated dose of iberdomide alone or in combination with dexamethasone in RRMM. For exploratory analysis, bone marrow aspirate clots were collected at both baseline and on cycle 2 day 15 (C2D15) and analyzed by immunohistochemistry (IHC) . There wde) Fig. , consistde) Fig. . The phade) Fig. . Cumulatapy Fig. . These rOverall, iberdomide biochemical potency translates into greater anti-MM activity than lenalidomide or pomalidomide in both IMiD-sensitive and -resistant MM cell lines Fig. S. These rSupplemental Methods and Figures


Oncolytic adenoviruses (Ad) have shown promising results in the therapeutic treatment of cancer. Ad type 5 (Ad5) is the most extensively utilized Ad type. However, several limitations exist to using Ad5 as an oncolytic virus, including high levels of anti-Ad5 neutralizing antibodies in the population, binding of the Ad5 hexon to blood coagulation factor X leading to liver sequestration and toxicity, and reduced expression of the primary receptor CAR on many tumors. Here, we use in vitro methods to explore the oncolytic potential of four alternative Ad types  belonging to the species D Ad subgroup and developed replication-competent species D Ads expressing the human sodium iodide symporter protein (hNIS) for combination radiovirotherapy. We evaluated the species D Ad vectors transduction, replication, cytotoxicity, and gene expression in six different cancer cell lines. Species D Ads showed the greatest transduction and cytotoxic killing in the SKBR3 breast cancer cells, followed by 293, A549, and HepG2 cells, however the cytotoxicity was less than the wild type Ad5 virus. In contrast, species D Ads showed limited transduction and cytotoxicity in the Hela and SKOV3 cancer cell lines. These species D Ad vectors also successfully expressed the hNIS gene during infection leading to increased iodide uptake in multiple cancer cell lines. These results, the low seroprevalence of anti-species D antibodies, and the lack of binding to blood coagulation FX, support further exploration of species D Ads as alternative oncolytic adenoviruses against multiple types of cancer. Oncolytic virotherapy, or the use of replication-competent viruses with a lytic life cycle to kill cancer cells, has promising therapeutic anticancer potential . Human aSpecies D Ads show many promising advantages that support their potential use in oncolytic virotherapy. We have specifically selected Ad26, 28, 45, and 48 from the species D Ad group because all four types have previously shown oncolytic activity against B-cell lymphomas ,17. Addi131I  (ATCC® HTB-77™), Hep G2 [HepG2] (ATCC® HB-8065™), SK-BR-3 [SKBR3] (ATCC® HTB-30™) were purchased from ATCC. Human kidney 293 cells were courtesy of Frank Graham . All cell lines were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin and incubated at 37 °C in a 5% CO2.A549 .The recombinant adenovirus genomes were released from the plasmid backbone through digestions with PacI. Plasmids were buffer-exchanged with StrataPrep PCR purification kit  and transfected into 6-well plates of 293 cells using Polyfect Transfection reagent . Cells were monitored for plaque formation and infected cells were subjected to three freeze–thaw cycles. Virus was amplified though sequential passage up to a Corning 10-cell stack flask . Virus was purified through two sequential CsCl gradients and a desalting column, Econo-Pac 10DG Desalting Columns . The virus particle quantity was determined by OD5 cells/well. Cells were infected the next day with 500 virus particles/cell (vp/cell) of the indicated replication-competent species D Ad-GFP-Luc virus or replication-defective Ad5-GFP-Luc viruses. Transduction was evaluated at 24 h to eliminate potential differences from replication profiles. GFP expression was examined 24 h post-infection and cells were imaged with an EVOS FL Cell Imaging System (Thermo Fisher). GFP images were overlayed with DAPI images using Image J to visualize cell confluency. Transduction was quantified by harvesting and fixing cells 24 h post infection with Ad-GFP-Luc viruses and then counting GFP+ cells on a BD Accuri™ C6 Plus Flow Cytometer. FX binding was evaluated by preincubating Ad-GFP-Luc virus with media containing either buffer or physiological levels of blood coagulation factor X for 10 min at 37 °C  before infecting confluent HepG2 cells at 500vp/cell and evaluating transduction by flow cytometry as described above.To examine transduction, cell lines were seeded in a 12-well plate at 2 × 104 cells/well and infected the next day with serial dilutions of the indicated wild type or Ad-hNIS virus ranging from 105 to 100 vp/cell. Five days post infection, 25 µL of MTT (4 mg/mL) was added to each well and incubated for 1 h at 37 °C to allow for formation of intracellular punctate precipitate. Media was then removed and 100 uL of acidified isopropanol solution (0.1 M HCl and 5% Triton X-100) was added before being read on a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices) at OD570 nm. Percent cell viability was determined by comparison to uninfected wells of each cell line. The EC50 was determined by fitting a sigmoidal dose–response curve using the GraphPad Prism software.Cell lines were seeded in a 96-well assay plate  at 2 × 105 cells/well and infected the next day with 500 vp/cell of the indicated Ad-hNIS virus. Total DNA was isolated from the cells at 24 and 72 h post-infection using the DNAeasy Blood and Tissue Kit . Real time PCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) and run on the QuantStudio 3 Real-Time PCR System (Applied Biosystems) using the following conditions: 50 °C for 2 min, 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, followed by a melt curve. Samples were run with Ad hexon primers Ad26/48 qPCR-F (5′-ACCGCCAGAGAACGCGCGAAGATGGCCACCC-3′) and Ad26/48 qPCR-R (5′-AGGCTGAAGTACGTGTCGGTGGCGCGGGC-3′) and normalized to levels of β-actin using primers ACTB gDNA qPCR-F (5′-GGCCTTGGAGTGTGTATTAAGT-3′) and ACTB gDNA qPCR-R (5′-GGACATGCAGAAAGTGCAAAG-3′). The results were compared to a standard curve created by dilution of pCR8 vector (Addgene) containing the Ad hexon fragment or β-actin gene and then results were converted to number of copies per cell.To examine Ad replication by qPCR, cell lines were seeded in a 12-well plate at 2 × 104 cells/well and infected the next day with 500 vp/cell of the indicated Ad-hNIS or Ad-GFP-Luc virus. At 48 h post infection, media was replaced with 10 µM of NaI and incubated for 3 h to allow for NaI uptake. After 3 h, cells were washed once with DPBS and 100 uL of ddH20 was added to each well followed by 100 uL of 10.5 mM ammonium cerium (IV) and 100 uL of 24 mM sodium arsenite (II). After 30 min incubation, the plate was read on a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices) at OD420 nm. NaI uptake was determined by comparison to a standard curve created by dilution of NaI.Sodium iodide symporter function was determined using a nonradioactive iodide uptake assay . Cell lip-value < 0.05 was considered statistically significant .GraphPad Prism software was used to analyze all data. Experiments were repeated in triplicate and graphed data represent the means of data from all experimental replicates with error bars representing standard deviation. A We developed four replication competent species D adenovirus (Ad) vectors to evaluate their oncolytic potential in the treatment of cancer. The wild type genomes of Ad26, 28, 45, and 48 were each cloned into a single low copy expression plasmid as previously described ,33. The First, we examined the ability of these species D Ads to transduce various cancer cells and compared this transduction to Ad5 as a benchmark. Our study utilized six cell lines from multiple different tissues to characterize the potential oncolytic activity against multiple cancer types . We usedOne major limitation to using Ad5 as an oncolytic virus is the strong binding of the Ad5 capsid hexon protein to blood coagulation factor X (FX). This Ad5-FX complex increases hepatocyte transduction through interaction with heparin sulfate proteoglycan on the hepatocytes and results in liver sequestration and toxicity ,35. In cNext we evaluated the ability of these species D Ads to result in cellular lysis and cell death of cancer cells . To evalCytotoxic killing was detected in all cell lines for all species D vectors, however the cytotoxicity was less than that of the wild type Ad5 virus A. NevertAfter evaluating the cytotoxic activity of the wild type viruses, we next determined the EC50 of the recombinant species D viruses expressing hNIS using the same methods . These rNext, we evaluated the replication capability of the species D Ad-hNIS viruses in each cancer cell line by using quantitative polymerase chain reaction (qPCR) to examine genomic amplification over time. DNA was isolated from Ad-hNIS infected cells at 24 and 72 h post infection for detection of Ad gDNA by qPCR. All four species D Ad vectors showed significant genomic amplification between the 24 and 72 h timepoints , indicatLastly, we determined the iodide uptake after infection with the Ad-hNIS vectors to confirm successful expression of a functional hNIS protein. A nonradioactive iodide uptake assay was performed 48 h post infection with an Ad-hNIS or Ad-GFP-Luc control virus . All fouHuman Ad5 is the most extensively used oncolytic Ad type in the treatment of cancer. However, the high levels of anti-Ad5 neutralizing antibodies in the population, significant liver sequestration and toxicity from Ad5 hexon binding to blood coagulation FX, and reduced expression of the primary receptor CAR on many tumors limits the anticancer efficacy of this type. The need for alternative Ad types in oncolytic treatments promoted us to investigate species D Ads, which show low seroprevalence rates in the population and a lack of species D Ad hexon binding to blood coagulation FX. Here, we investigated the oncolytic potential of four species D Ads  using in vitro methods and develop a replication-competent species D Ads expressing hNIS for combination radiovirotherapy.First, we evaluated transduction efficiency in six different cancer cell lines and found that all species D Ads were able to transduce SKBR3, 293, A549, and HepG2, but only weakly transduce the Hela and SKOV3 cell lines. Notably, the species D Ads transduced SKBR3 and HepG2 cells at high levels similar levels to the Ad5 virus. Importantly, the primary cellular receptor usage for these species D Ads has not been fully determined and remains controversial in the literature. For example, some studies have suggested that Ad26 uses CD46 as a cellular receptor ,22,23 whImportantly, unlike the Ad5 virus, preincubation of species D Ads with blood coagulation FX did not result in increased transduction of hepatocytes. This data supports the previous finding that the hexon capsid protein of species D Ads does not bind to blood coagulation FX and therefore does not exhibit liver sequestration and toxicity ,11. BiodSpecies D Ads have previously been explored for their increased transduction and oncolytic potential against B cell cancers ,17, and 131I [Finally, expression of the hNIS gene by the oncolytic species D Ads resulted in cellular iodide uptake and concentration, with the strongest concentration observed in the SKBR3 cancer cell line. Expression of the hNIS gene by these species D Ads could enhance the anticancer potential by improving tumor targeting through noninvasive nuclear imaging or by direct cell killing from intracellular concentration of radioactive 131I . HoweverHere, we evaluated oncolytic potential of four species D Ad types  in vitro. We found that the species D Ad vectors showed the most promising cytotoxic activity and gene expression in the SKBR3 breast cancer cells, followed by 293, A549, and HepG2 cell lines, with limited activity in Hela and SKOV3 cells. Combined with radiovirotherapy, these species D vectors could provide a dual mechanism for targeting several carcinomas, especially in cancers that are refractory to standard treatment. Studies evaluating oncolytic efficacy, biodistribution, and radiovirotherapy treatments in vivo would further support the therapeutic activity of these species D viruses. Excitingly, the initial in vitro results of this study, paired with previous evidence of low seroprevalence and lack of liver toxicity, supports the continued development and characterization of these species D Ads as alternative oncolytic adenoviruses against multiple types of cancer.Here, we evaluated the oncolytic potential of four species D Ad types . Although the cytotoxicity of these species D Ads was less than Ad5, these species D Ad vectors still showed promising transduction, cytotoxic activity, and gene expression in the SKBRS, 293, A549, and HepG2 cell lines. The initial in vitro results of this study, paired with previous evidence of low seroprevalence and lack of liver toxicity, supports the continued development and characterization of these species D Ads as alternative oncolytic adenoviruses against multiple types of cancer.


Klotho was first discovered as an anti-ageing protein linked to a number of age-related disease processes, including cardiovascular, renal, musculoskeletal, and neurodegenerative conditions. Emerging research has also demonstrated a potential therapeutic role for Klotho in cancer biology, which is perhaps unsurprising given that cancer and ageing share similar molecular hallmarks. In addition to functioning as a tumour suppressor in numerous solid tumours and haematological malignancies, Klotho represents a candidate therapeutic target for patients with these diseases, the majority of whom have limited treatment options. Here, we examine contemporary evidence evaluating the anti-neoplastic effects of Klotho and describe the modulation of downstream oncogenic signalling pathways, including Wnt/β-catenin, FGF, IGF1, PIK3K/AKT, TGFβ, and the Unfolded Protein Response. We also discuss possible approaches to developing therapeutic Klotho and consider technological advances that may facilitate the delivery of Klotho through gene therapy.  Together, these activate FGF receptor 1c (FGFR1c) in the kidney to stimulate phosphate wasting. Outside of phosphate regulation, the FGF pathway is also involved in diverse biological events such as cell differentiation, migration and proliferation, and Klotho may play a central role in modulating these pathways  Figure a. Wolf e2+ response and significantly decrease cell proliferation and clonogenicity. In addition, increased apoptosis induced by cytotoxic endoplasmic reticulum (ER) stressors such as gemcitabine, thapsigargin, and ABT-737 was observed. The authors also demonstrated that the afore-described cellular effects could be counteracted by IGF-1R-dependent signalling and activate Ca2+-dependent ER stress. Furthermore, the mechanism by which Klotho caused cell death in this LPS model was shown to be related to increased reticular Ca2+-leakage.IGF-1R is a receptor tyrosine kinase that stimulates proliferation by regulating downstream targets, including IRS proteins, the PI3K-AKT-TOR and RAF-mitogen-activated protein kinase (RAF-MAPK) pathways  Figure a. High lThe PI3K/Akt is an important downstream component of the IGF-1R pathway that regulates gene transcription, cell cycle events, and cell proliferation  Figure a. In a sMoreover, Dalton et al. undertook a study to better understand the mechanism by which sKL acts to mediate IGF-1 driven PI3K/Akt signalling . Using HAPC gene, which functions as a negative regulator of β-catenin is mutated in up to 85% of sporadic colon cancers, emphasising the importance of targeting the WNT pathway in this tumour type [Aberrant activation of the WNT signalling pathway is associated with the development of numerous human cancers, including colorectal and HCC . This hiour type . Finallyour type ,102.TGF-β signalling plays a biphasic role in the development and progression of cancer ,104,105.The UPR is a complex of signalling pathways that are able to maintain a productive endoplasmic reticulum (ER) protein-folding environment b. These Stanniocalcin (STC), a secreted glycoprotein hormone involved in calcium and phosphate homeostasis  that is Despite the considerable interest in Klotho since its discovery over two decades ago, no Klotho-based therapies have reached clinical trials for a variety of reasons. Several strategies focused on delivering, restoring, or modulating Klotho levels have been considered. Delivery of the recombinant full-length Klotho protein due to its inherent large molecular weight and potential instability is challenging. One reasonable approach would be to consider information derived from the available structures of Klotho ,124,125.The identification of molecules that could modulate Klotho levels or Klotho-dependent signalling has been of interest, particularly for the treatment of neurodegenerative conditions or neurological malignancies where the introduction of an exogenous Klotho protein into the brain is impractical due to its large molecular weight and inability to cross the blood-brain barrier . MoreoveGene therapy, defined as the introduction of genetic material into a target cell for therapeutic benefit, represents an alternative approach to treating disease . To dateDNA methylation is a covalent modification of chromatin that is critical for regulating biological processes such as cellular differentiation and maintenance of cellular homeostasis ,145,146.Klotho was initially discovered as an anti-ageing protein but has since generated considerable interest due to its potential therapeutic role in cancer biology, which is perhaps unsurprising given that cancer and ageing share similar molecular hallmarks. Studies comparing normal and tumour tissues have illustrated that the downregulation of Klotho is associated with cancer development and worsening of patient outcomes. This has highlighted Klotho’s potential role as a tumour suppressor, as well as a prognostic tumour biomarker with the potential to aid the early detection of malignancies. In addition, Klotho over-expression generally leads to decreased cancer cell viability, and treatment with soluble Klotho has resulted in reduced tumour volume in pre-clinical cancer models, including stomach, pancreas, colon, and breast. However, there are challenges that lie ahead in the advancement of Klotho as a therapeutic target. First, the exact mechanism by which Klotho exerts its anti-tumour effects is poorly understood and likely to be context-dependent. The abrogation of the diverse biological pathways that signal downstream of Klotho have been shown in several cancer models, as discussed here. However, the ligand-binding domains of Klotho responsible for downstream pathway modulation are not fully characterised, and the observation that Klotho binds to monosialogangliosides to regulate membrane microdomains opens up the possibility that pathway alterations via these gangliosides may be the critical link in deciphering the anti-neoplastic effects of Klotho . Secondl


Long noncoding RNA DiGeorge syndrome critical region gene 5 (DGCR5) has been shown to be highly associated with cancer development. However, the biological role and molecular mechanism of DGCR5 in pancreatic cancer (PC) remains largely unknown. This study aimed to explore the role of DGCR5 in PC. It was revealed that DGCR5 was highly expressed in PC tissues compared with adjacent normal tissues and was associated with poor prognosis in PC patients. Furthermore, DGCR5 depletion inhibited the proliferation, migration and invasion by increasing apoptosis and inducing G0/G1 cell cycle arrest in vitro. Moreover, xenograft assay validated that DGCR5 promotes PC tumor growth in vivo. Mechanistically, DGCR5 was found to act as a ceRNA by sponging miR-3163 to regulate DNA topoisomerase 2-alpha (TOP2A) and inhibit Wnt/β-catenin pathway. In addition, it was found that DGCR5 downregulation could enhance the sensitivity of PC cells to gemcitabine, and ChIP assay showed that PAX5 (Paired Box 5) could bind to the promoter region of DGCR5 and increase its transcription. The results of the present study indicated that DGCR5 may be a potential diagnostic biomarker and therapeutic target for PC. Pancreatic cancer (PC), one of the most rapidly lethal tumors, is the fourth most common cause of cancer mortality worldwide with the least patient survival rate of any cancer Data from the human whole genome and transcriptome studies revealed that more than 90% of the genome is transcribed into RNA, but less than 2% of them encodes proteins, whereas the majority of the malignant genome encodes a large number of noncoding RNAs (ncRNAs) In the present study, we found that DGCR5 was overexpressed in PC tissues compared with adjacent normal tissues and its high expression was associated with poor prognosis in PC patients. Knockdown of DGCR5 inhibited the proliferation, migration and invasion by inducing cell apoptosis and G0/G1 cell cycle arrest of PC cells in vitro, and suppressed PC tumor growth in vivo. Mechanistically, DGCR5 functions as a competing endogenous RNA (ceRNA) by sponging miR-3163 to regulate TOP2A and inhibit Wnt/β-catenin pathway. Moreover, we found that DGCR5 downregulation enhanced the sensitivity of PC cells to gemcitabine, and DGCR5 was activated by the transcription factor PAX5. All these findings demonstrated that DGCR5 may serve as a potential diagnostic biomarker and therapeutic target for PC.Specimens were obtained from 38 patients with a pathological diagnosis of PC who had neither chemotherapy nor radiotherapy before surgical resection, between 2015 and 2017 at the Department of General Surgery, Xinhua hospital, School of Medicine, Shanghai Jiaotong University, China. This study was approved by the Ehics Committee of Xinhua Hospital of Shanghai Jiaotong University School of Medicine.2 humidified incubator, and cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco).Human PC cell lines , and immortalized human non-cancer pancreatic cell line (HPNE) were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences . Cells were maintained at 37℃ in a 5% CODGCR5 small interfering RNAs (siRNAs), hsa-miR-3163 mimics and inhibitor, and their parental negative control (NC) were synthesized by Genomeditech  and transfected into cells using RFect reagent  according to the manufacturer's protocol. The siRNA sequences are: si-DGCR5-1: 5'-GCAAUUAGCUUCAGCUCUA-3', si-DGCR5-2: 5'-GCGAGAUGUUAUUUCUGAA-3', si-DGCR5-3: 5'-GGUGGUGCACGAGAGUCUA-3'; si-TOP2A: 5′-CUAUUCAUCUUUCCCGAGA-3′; si-PAX5-1: 5'-GAGGAUAGUGGAACUUGCUCAUCAA-3', si-PAX5-2: 5'-GGTAATTGGAGGATCCAAA-3'. Lentiviruses expressing full-length of DGCR5, sh-DGCR5 (5'-GCAAUUAGCUUCAGCUCUAdTdT-3') and NC were constructed by Genomeditech . Cells were infected at a multiplicity of infection (MOI) of 90 in complete culture medium for 48 hours, and then subjected to selection with 1μg/ml puromycin for 1 week to construct stable-transfected cells. Transfection efficiency was verified by quantitative real-time PCR (qRT-PCR).-ΔΔCT method was used for the quantification of gene expression. The primer sequences were synthesized by HuaGene Biotech  and listed as followed: DGCR5 forward: 5ʹ-ATTTTCCCAGTCTGGCGGAG-3ʹ, reverse: 5ʹ-AGGGCCCCATTATGACTCCT‐3ʹ; TOP2A forward: 5ʹ-ACCATTGCAGCCTGTAAATGA-3ʹ, reverse: 5ʹ-GGGCGGAGCAAAATATGTTCC‐3ʹ; GAPDH forward: 5′-CAACAGCCTCAAGATCATCAGC-3′, reverse: 5′-TTCTAGACGGCAGGTCAGGTC-3′; PAX5 forward: 5'-ACTTGCTCATCAAGGTGTCAG-3'; reverse: 5'-TCCTCCAATTACCCCAGGCTT-3'; U6 forward: 5'-AAAGCAAATCATCGGACGACC-3', reverse: 5'-GTACAACACATTGTTTCCTCGGA-3'.Total RNA was isolated by TRIzol reagent according to the manufacturer's instructions. The Mir-X miRNA qRT-PCR TB Green Kit (Takara) was used for converting miR-3163, other cDNAs were generated by PrimScript Reverse Transcriptase (Takara) according to the manufacturer's instruction. QRT-PCR was performed using SYBR Premix Ex Taq II (Takara) on StepOnePlusTM Real-time PCR system . 2Cellular fractionation of DGCR5 was measured using DGCR5 FISH probes and kit designed and synthesized by GenePharma  following the manufacturer's instructions.Cells were plated into a 96-well plates overnight at the density of 2000 cells/well with complete culture medium DMEM. Each well was replaced with 10 μl CCK-8 and 90 μl complete medium. After incubating for 2 hours at 37℃ away from light, optical density (OD) value at 450nm was detected through a microplate reader (Bio-Tek).500 treated cells were plated in each well of a 6-well plate for 7 to 10 days. The cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. After washing and drying the plate, cell colonies were counted and analyzed.BeyoClickTM EdU-488 proliferation assay  was used to examine the effect of DGCR5 on cell proliferation according to the manufacturer's protocol. Cells were incubated with 10μM EdU for 2 hours, stained with Azide488 and Hoechst 33342 then visualized under a fluorescence microscope (Leica).4 cells with 200 μl serum-free medium were plated in the upper chamber. After incubation for 24 hours, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells remaining on the upper membrane were gently washed with PBS and scraped by cotton swabs. Stained cells on the bottom of the chamber were counted under a microscope on 5 random fields.Cell migration and invasion capabilities were measured using Transwell chamber inserts  and Corning BioCoat Growth Factor Reduced Matrigel Invasion Chambers , respectively. 750 μl DMEM with 10% FBS was added into the lower chamber, 3 × 10Cells in apoptosis were detected using an Annexin V/PI apoptosis assay kit . In brief, the transfected cells were collected and re-suspended with PBS, stained with 5 μl Annexin V-FITC and/or 5 μl PI for 30 minutes at room temperature shielded from light. Cell apoptosis was immediately determined by flow cytometry.For cell cycle analysis, transfected cells were harvested and fixed with 75% ethanol at 4℃ overnight. After fixation, cells were washed and re-suspended with PBS and incubated with 10mg/ml RNase and 1mg/ml PI at 37 ℃ for 30 minutes in the dark. The cell cycle was analyzed by flow cytometry.Protein was extracted from transfected cells with RIPA lysis buffer. Equal amounts of protein samples were loaded and separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk in TBST for 1 hour at room temperature. Then, membranes were incubated with diluted primary antibodies at 4℃ overnight. The antibody against PAX5 was obtained from Abcam, other antibodies were acquired from Cell Signaling Technology. Next, membranes were rinsed with TBST 3 times, followed by incubating with secondary antibody for 1 hour, rinsed again with TBST and finally determined by a Gel Doc 2000 (Bio-Rad).7 in 200 μL PBS) transfected with LV-NC (2 groups), LV-shDGCR5 or LV-DGCR5 were injected hypodermically into the left axilla of the mice. Starting from Day 7, tumor volumes were estimated every 7 days by measuring the tumor length and width by caliper. Tumor volume was calculated as π/6 × width2 × length. All mice were sacrificed by dislocation after 4 weeks, tumors were collected for further assays. The study was approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine.Nude mice  with 18-22g were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences . In this assay, nude mice were randomly divided into 4 groups (n = 5 per group). PaTu8988 cells , and the level of expression was assessed by Image J software.Total RNA from NC or DGCR5-knockdown Mia PaCa-2 and PaTu8988 cells was extracted and subjected to mRNA sequencing on the BGISEQ-500 platform at the Beijing Genomics Institute . The differentially expressed genes in both cell lines were selected that met the criteria of a fold change ≤ -1.0 or ≥ 0.85 and P ≤ 0.05 after DGCR5 knockdown. The analysis were performed using the BGI online analysis system.http://carolina.imis.athena-innovation.gr) and TargetScan (http://www.targetscan.org) databases were used to search the potential miRNA that interacts between DGCR5 and TOP2A. The transcription factor PAX and its binding sites in the promoter region of DGCR5 was identified by PROMO , GeneCards (http://genecards.org) and JASPAR (http://jaspar.genereg.net/).The DIANA-LncBase , miR-3163 mimics, NC mimics, miR-3163 inhibitor or NC inhibitor. After 48 hours of incubation, the relative luciferase activities were determined by Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol.293T cells were plated at 2 × 10ChIP assays were performed with Mia PaCa-2 and PaTu8988 cells via ChIP Assay Kit  following the manufacturer's instruction. Anti-PAX5 antibody and IgG antibody were obtained from Abcam. The ChIP samples were analyzed via qRT-PCR and the PCR products were verified by 2% agarose gel electrophoresis. The primer sequences of PAX5-DGCR5 are as follow: forward: 5'-TGCTTGTTTAGGGGTCAGGC-3'; reverse: 5'-CTGAGGTGGCTCAACAGGTC-3'.Statistical analyses were performed using GraphPad Prism 7 and the results were presented as mean ± standard deviations (SD). The student's t tests were performed for comparisons between 2 groups and one-way ANOVA was used for multiple group comparisons. The Chi-square test was conducted to compare clinicopathological features of the patients with DGCR5 expression. The Kaplan-Meier plots and log-rank tests were used for the overall survival analysis. The multivariate cox proportional hazards analysis was applied for the multivariate survival analysis. The correlation between DGCR5 and TOP2A was analyzed by Spearman's correlation test. Data were presented as mean ± SD of at least three independent experiments. Results with P < 0.05 were considered statistical significant.p< 0.05). As expected, DGCR5 was overexpressed in PC cell lines  compared with normal human pancreatic cell (HPNE)  in Mia PaCa-2 and PaTu8988 cells after DGCR5 knockdown were selected, including 49 downregulated genes and 12 upregulated genes (Figure In addition, we found that DGCR5 overexpression increased the expression of TOP2A, and TOP2A knockdown inhibited the cell proliferation that promoted by DGCR5 upregulation Figure H.It is widely known that lncRNAs can act as ceRNAs to regulate mRNA in the cytoplasmAs we know, the Wnt/β-catenin signaling pathway is particularly relevant in PC and it has been reported that TOP2A promoted malignant progresssion of PC through activating this pathwayIn summary, the above data verified that DGCR5 regulates the PC progression by sponging miR-3163 to target TOP2A and activating Wnt/β-catenin pathway.Our previous data has proved TOP2A as a downstream gene of DGCR5, which was found associated with gemcitabine resistance in PC patients by gene expression analysisTo further investigate the transcriptional regulatory mechanism of DGCR5 in PC, we search for the transcription factors that may regulate DGCR5 by performing bioinformatics analysis using PROMO, GeneCards and JASPAR databases. PAX5 was identified from the candidate transcription factors for its high predicted scores in all databases, and the predicted binding sites of PAX5 in the promoter sequence of DGCR5 are shown in Figure There is increasing evidences that lncRNAs exert important roles and to act through a variety of molecular mechanisms, such as ceRNAs, in the development and progression of various cancers, including PC We applied loss-of-function and gain-of-function approaches and demonstrated that DGCR5 performed significant effects on the biological processes of PC. Here, we validated DGCR5 was notably upregulated in PC tissues and cell lines, and DGCR5 knockdown markedly suppressed PC proliferation, DNA replication, migration, invasion, EMT process, and induced cell apoptosis and G0/G1 cell cycle arrest in vitro. Similarly, the tumor xenograft model assays displayed that DGCR5 enhanced the tumorigenicity of PC in vivo.In general, the function of lncRNAs are mainly determined by its subcellular localization, and lncRNAs located in the cytoplasm always exert effects through the regulatory mechanism of ceRNA by sponging specific miRNAs to restore target gene expression To complete the ceRNA network of DGCR5, we performed bioinformatics analysis and miR-3163 was predicted to be the link between DGCR5 and TOP2A. We first examined the relation of the expression of DGCR5, miR-3163 and TOP2A, then conducted dual-luciferase assays and proved that miR-3163 could directly bind to DGCR5 and TOP2A. These results suggest the DGCR5/miR-3163/TOP2A axis as a regulatory mechanism in PC.Wnt/β-catenin pathway has been demonstrated to play an important role in PC progression, therefore, we focused on this signaling pathway to characterize the molecular mechanisms of DGCR5 in mediating PC progression. We determined the expression of several key regulators by IHC staining and western blot, and found that DGCR5 activated the pathway, whereas TOP2A knockdown suppressed the activation induced by DGCR5. Our data suggest that DGCR5/miR-3163/TOP2A axis mediates pancreatic carcinogenesis by modulating wnt/β-catenin pathway.Gemcitabine-based treatment is the most widely used chemotherapy for PC patients, however, chemoresistance remains a major obstacle to its clinical success Recent studies manifested that the transcription factor PAX5 is highly involved the regulatory network of human malignancy as an oncogene via its transactivation function In summary, we characterized the role of DGCR5 in PC for the first time, that DGCR5 is activated by PAX5 and promotes PC progression by sponging miR-3163 to regulate TOP2A and activating wnt/β-catenin pathway Figure F. ConseqSupplementary figure.Click here for additional data file.


Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs.klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes.In an attempt to produce Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.The online version contains supplementary material available at 10.1186/s12896-020-00660-9. Genetically engineered pigs are useful models for studying human diseases due to the similarity of their anatomy and physiology to those of humans . Recent CRISPR-mediated genome editing in pigs is generally accomplished by somatic cell nuclear transfer (SCNT) with donor cells transfected with single-guide RNA (sgRNA) and Cas9 DNA . Howeverklotho gene expression display multiple aging-like phenotypes similar to human premature-aging syndromes .Total RNA was extracted using the easy-spin Total RNA Extraction Kit , according to the manufacturer’s protocol, and the total RNA concentration was quantified using a NanoDrop 2000 Spectrophotometer . The cDNA was synthesized using Maxime RT Premix (iNtRON) according to the manufacturer’s protocol. A PCR plate  was made by adding 1 μL cDNA, 0.4 μL (10 pmol/μL) forward primer, 0.4 μL (10 pmol/μL) reverse primer, 10 μL SYBR Premix Ex Taq  and 8.2 μL of Nuclease-free water . The reactions were carried out for 40 cycles and the cycling parameters were as follows: denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min and extension at 72 °C for 1 min. All oligonucleotide primer sequences are presented in Table Fetal fibroblasts and placentas were finely grinded by homogenizer and washed with PBS and lysed with ice-cold lysis buffer . The lysate was clarified by centrifugation . Equal amounts of proteins from the supernatant was fractionated by 10% SDS-PAGE and electrotransferred onto a PVDF membrane. The blots were blocked in 5% skim milk at 4 °C overnight. In the next day, the membrane was incubated with rabbit anti-klotho antibody  and rabbit anti-β-actin  diluted with 5% skim milk Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h. Then membranes were washed three times each for 10 min with TBST incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000) for 1 h. The blots were developed using the Pierce SuperSignal West Pico Chemiluminescent System (Thermo Fisher Scientific). The densities of the immunoblots were scanned with image acquisition system .klotho gene were predicted using the Cas-OFFinder online tool (http://www.rgenome.net/cas-offinder/) to analyze site-specific cleavage by the CRISPR/Cas9 system. All the potential OTSs were PCR amplified and their PCR products were sequenced using an ABI 3730XL DNA analyzer  to confirm the off-target events. Sequencing data were analyzed using a Variant Reporter Software Version 2.0 (Applied Biosystems). Sequences of the potential OTSs are listed in Additional file Ten potential off-target sites (OTSs) for the sgRNA targeting porcine U-test for data with non-normal distribution or Student’s t-test for data with normal distribution to determine differences between experimental groups. Data are expressed as means ± SEM. P values < 0.05 were considered to be statistically significant.Statistical analyses were performed using SPSS 22.0 . All data were tested for normality and homoscedasticity, then subjected to a Mann-Whitney’s Additional file 1: Table S1. Summary of potential off-target sites (OTSs) for the sgRNA targeting porcine klotho gene. Table S2. Primer sequences of potential off-target sites (OTSs) for the sgRNA targeting porcine klotho gene.Additional file 2: Figure S1. Uncropped gel images for Fig. Figure S2. Uncropped immunoblot images for Fig. klotho monoallelic knockout and wild-type placentas detected by Western blot analysis. WT, wild-type; Fetus V2; viable fetus 2 . Figure S3. Uncropped immunoblot images for Fig. klotho monoallelic knockout and wild-type placentas detected by Western blot analysis. WT, wild-type; Fetus V2; viable fetus 2 .