Transfected Cell Lines Research Articles

Exosomes derived from cardiac progenitor cells attenuate CVB3-induced apoptosis via abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways

Viral myocarditis is potentially fatal and lacking a specific treatment. Exosomes secreted by cardiac progenitor cells (CPCs) have emerged as a promising tool for cardioprotection and repair. In this study, we investigated whether CPCs-derived exosomes (CPCs-Ex) could utilize the mTOR signal pathway to reduce the apoptosis in viral myocarditis. In vitro, exosomes were, respectively, added to H9C2 cells after CVB3 infection to detect the anti-apoptosis effect of CPCs-Ex. Compared with the controls, the apoptosis rate was reduced, accompanied with the depressed expression of viral capsid protein 1 (VP1) and pro-apoptosis factors of Bim/caspase families. Meanwhile, the phosphorylation of Akt, mTOR, and p70S6K were promoted, but that of 4EBP1 was suppressed. In vivo, the results of apoptosis, expression of CVB3 and pro-apoptosis factors, and phosphorylation of Akt/mTOR factors of CVB3-infected cardiomyocytes were consistent with that of vitro. Following that, we use Rapamycin and MK-2206 to inhibit the Akt/mTOR signaling pathway, meanwhile, Rattus 4EBP1, p70S6K, Akt1 and Akt2 were transfected to H9C2 cells to establish the stably transfected cell lines. In the group with Rapamycin or MK-2206 pretreatment, CPCs-Ex also could decrease the apoptosis of H9C2 cells and expression of CVB3 mRNA, followed by decreased expression of apoptosis factors. In Akt2, p70S6K and 4EBP1 overexpression groups, CPCs-Ex promoted CVB3-induced apoptosis, VP1 expression and cleavage of caspase-3. Our results therefore identify CPCs-Ex exerts an anti-apoptosis effect in CVB3-infected cells by abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways as well as the expression of Bcl-2 and caspase families. Viral myocarditis, mainly caused by CVB3 infection, is lacking a specific treatment. Our study identified an anti-apoptosis role of CPCs-Ex in CVB3-infected cells and rats, which shown that CPCs-Ex may be an effective tool to treat viral myocarditis. We believe that with more in-depth research on the functionality of CPCs-Ex, there will be a breakthrough in the treatment of viral myocarditis.

Long Noncoding RNA GAS5 Acts As A Tumor Suppressor In Laryngeal Squamous Cell Carcinoma Via miR-21.

PurposeLong noncoding RNAs (lncRNAs) have been identified as an important class of noncoding RNAs that are deeply involved in multiple biological processes in tumorigenesis. This study is to investigate the critical roles and biological function of lncRNA growth arrest-specific 5 (GAS5) in tumorigenesis of laryngeal squamous cell carcinoma (LSCC).Patients and methodsA total of 59 samples of LSCC and paired adjacent tissue, as well as corresponding clinicopathological information were collected. GAS5 expression in both LSCC tissues and human SUN1076 and SNU899 cell lines were analyzed by Real-time quantitative RT-PCR method. Ectopic expression of GAS5 by vector transfection in LSCC cell lines and followed by in vitro experiments was to investigate the critical roles and function of GAS5 in LSCC. Cell Counting Kit 8 (CCK8) assay and PE/7AAD Annexin V Apoptosis analysis was to evaluate cell proliferation ability and cell apoptosis. Co-transfection of GAS5 and miR-21 was to explore the interaction between GAS5 and miR-21 in LSCC. BAX and CDK6 protein level were analyzed by western blot method.ResultsThis study demonstrated that GAS5 was significantly downregulated in LSCC tissue and human LSCC cell lines. GAS5 levels were correlated with the clinicopathological features of LSCC patients. In addition, the ectopic expression of GAS5 significantly inhibited cell proliferation and promoted apoptosis. Co-expression analyses indicated that GAS5 is negatively correlated with miR-21 in LSCC tissues. Overexpression of miR-21 eliminated GAS5-mediated cell apoptosis and proliferation suppression. Furthermore, GAS5, which upregulated BAX mRNA expression and downregulated CDK6 mRNA expression, was reversed by ectopic expression of miR-21.ConclusionGAS5 suppresses LSCC progression through the negative regulation of miR-21 and its targets involved in cell proliferation and apoptosis, indicating that GAS5 may serve as a biomarker and potential target for LSCC therapy.

Long noncoding RNA LINC00511 involves in breast cancer recurrence and radioresistance by regulating STXBP4 expression via miR-185.

The aim of this study was to explore the molecular function of long intergenic noncoding RNA 00511 (LINC00511) and its target proteins in recurrent breast cancer after breast-conserving surgery followed by radiotherapy. LINC00511 expression in tissues was measured by quantitative polymerase chain reaction (qPCR). The association between LINC00511 expression and the clinicopathological features of breast cancer was analyzed by Chi-square tests. The impact of LINC00511 on overall survival was evaluated by the log-rank test. MDA-MB-231/MDA-MB-436 cell lines transfected with short hairpin RNA (shRNA) were used to investigate the influence of LINC00511 silencing on tumor growth and radiosensitivity in vitro and in vivo. A series of experiments including cell apoptosis assay, cell colony formation assay, and mouse xenograft models were applied to test those transfected cell lines. MicroRNA (miRNA) targets of LINC00511 were identified by bioinformatics analysis and further validated by dual luciferase reporter assay, qPCR, and Western blot analysis. LINC00511 expression was significantly increased in breast cancer tissues and correlated with recurrence and poor survival after breast-conserving surgery followed by radiotherapy. LINC00511 knockdown by shRNA restricted cell proliferation, promoted cell apoptosis, and enhanced radiosensitivity in vitro, and inhibited tumor growth with an increased response to radiation in vivo. In addition, elevated LINC00511 was found to increase syntaxin-binding protein 4 (STXBP4) expression through competitive binding to miR-185, while silencing LINC00511 decreased STXBP4 expression and increased radiosensitivity. LINC00511 inhibition impairs its competitive binding to miR-185, resulting in increased STXBP4 expression and improved radiation response in breast cancer. Our study results suggest that the LINC00511/miR-185/STXBP4 axis may be a promising therapeutic target for improving the prognosis of breast cancer.

Biological roles of filamin a in prostate cancer cells.

ABSTRACTObjective This study aims to investigate the association of filamin A with the function and morphology of prostate cancer (PCa) cells, and explore the role of filamin A in the development of PCa, in order to analyze its significance in the evolvement of PCa.Materials and Methods A stably transfected cell line, in which filamin A expression was suppressed by RNA interference, was first established. Then, the effects of the suppression of filamin A gene expression on the biological characteristics of human PCa LNCaP cells were observed through cell morphology, in vitro cell growth curve, soft agar cloning assay, and scratch test.Results A cell line model with a low expression of filamin A was successfully constructed on the basis of LNCaP cells. The morphology of cells transfected with plasmid pSilencer-filamin A was the following: Cells were loosely arranged, had less connection with each other, had fewer tentacles, and presented a fibrous look. The growth rate of LNCap cells was faster than cells transfected with plasmid pSilencer-filamin A (P <0.05). The clones of LNCap cells in the soft agar cloning assay was significantly fewer than that of cells stably transfected with plasmid pSilencer-filamin A (P <0.05). Cells stably transfected with plasmid pSilencer-filamin A presented with a stronger healing and migration ability compared to LNCap cells (healing rate was 32.2% and 12.1%, respectively; P <0.05).Conclusion The expression of the filamin A gene inhibited the malignant development of LNCap cells. Therefore, the filamin A gene may be a tumor suppressor gene.

Expression and association of IL-21, FBXL20 and tumour suppressor gene PTEN in laryngeal cancer.

ObjectiveTo study the expression of three genes IL-21, FBXL20 and tumour suppressor gene PTEN in laryngeal cancer; analyse the differences in their expression in laryngeal cancer and adjacent tissues; by using pEGFP-N1-IL21 and pGPU/GFP/Neo-FBXL20 expression vectors, to analyse the characteristics in their expression in laryngeal cancer cells outside the body as well as the associations among them.MethodsThe expression of the three genes in laryngeal cancer and adjacent tissues from 30 cases and in normal laryngeal tissues from 20 healthy persons was detected with the RT-PCR; laryngeal cancer cell line (HEp-2 cells) transfection was also performed with the pEGFP-N1-IL21 and pGPU/GFP/Neo-FBXL20 expression vectors we constructed, to detect the mRNA expression of the three genes. Cell proliferation, apoptosis and cell cycle were measured by the MTT assay.ResultsThe results of RT-PCR showed that the expression of IL-21 and FBXL20 was up-regulated in laryngeal cancer, while the expression of tumour suppressor gene PTEN was significantly decreased (p < 0.01). In HEp-2 cells transfected with pGPU/GFP/Neo-IL-21 and pGPU/GFP/Neo-FBXL20 expression vectors, the mRNA expression of PTEN was restored to some extent (p < 0.05); in addition, the ability of HEp-2 cells in proliferation and invasion was also reduced.ConclusionsIL-21 and FBXL20 genes are important in the occurrence and development of laryngeal cancer; the expression of PTEN gene can suppress laryngeal cancer, and there's a certain association among IL-21, FBXL20 and PTEN.

In vitro evidence consistent with an interaction between wild-type and mutant SOD1 protein associated with canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis (fALS). DM is associated with a c118G>A mutation of the superoxide dismutase 1 (Sod1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS. The consensus view is that these Sod1/SOD1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild-type and mutant forms of SOD1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein (EGFP) tag. These fusion proteins retained SOD1 enzymatic activity on a native gel assay system. We demonstrate that SOD1 aggregate density is significantly higher in DM transfectants compared to wild-type. In addition, we show by co-immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild-type and mutant forms of SOD1 in co-transfected cells. While in vitro studies have shown SOD1 heterodimer formation in fALS models, this is the first report for DM SOD1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM, a similar interaction between wild-type and mutant SOD1 forms can occur. Clarifying the role of SOD1 in DM may also be of benefit to understanding the role of SOD1 in fALS.

Functional characterization of seven-band grouper immunoglobulin like cell adhesion molecule, Nectin4 as a cellular receptor for nervous necrosis virus

Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1 cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.

Ecdysteroid-mimicking compounds act as both agonists and antagonists to the crustacean ecdysone receptor

To characterize the potential endocrine-disrupting chemicals (EDCs) in the environment that interact with the crustacean ecdysone receptor (EcR), we established a method involving in silico modeling/molecular docking and in vitro reporter gene assay. Cherry shrimp (Neocaridina davidi) EcR (NdEcR) and retinoid X receptor (NdRxR) were identified and cloned for use in this method. A theoretical 3D model of NdEcR ligand-binding domain (LBD) was built in silico based on sequence homology with the established X-ray structure of insect EcR. The interaction of the NdEcR LBD with ecdysteroids, diacylhydrazine (DAH) pesticides, and other potential EDCs was evaluated using molecular docking programs. The results revealed that the ligand-binding pocket in the NdEcR LBD was flexible and adaptive for accommodating ligands of different shapes. The agonistic and antagonistic activities of the candidate compounds were further assessed by in vitro reporter gene assay using human cell lines transiently transfected with NdEcR and NdRxR expression plasmids and a reporter plasmid containing synthesized ecdysone response element. The assay was validated by the dose-dependent responses of EcR-mediated gene transcription after treating the transfected cell lines with ecdysteroids, 20-hydroxyecdysone, and ponasterone A. Examination of the candidate compounds using the reporter gene assay revealed restricted functional specificity to ecdysteroids and DAHs. Three of the tested DAH pesticides originally targeting the insect EcR were found to be weak agonists and strong antagonists of NdEcR. These results suggest that DAHs are potential EDCs for crustaceans that disrupt their ecdysteroid signals by functioning as EcR agonists or antagonists.

Mechanism of lncRNA FEZF1-AS1 in promoting the occurrence and development of oral squamous cell carcinoma through targeting miR-196a.

Previous studies have demonstrated that long non-coding ribonucleic acid (lncRNA) FEZF1-AS1 acts as a cancer-promoting gene. However, no reports have investigated the role of FEZF1-AS1 in oral squamous cell carcinoma (OSCC) yet. Therefore, the aim of this study was to explore whether FEZF1-AS1 promoted the expression characteristics of OSCC by targeting miR-196a and to further elucidate the underlying mechanism of FEZF1-AS1 in promoting the metastasis of OSCC. The expression levels of FEZF1-AS1 and miR-196a in 42 pairs of OSCC tissues and para-carcinoma tissues were detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation of FEZF1-AS1 expression with clinical indexes and prognosis of OSCC patients was analyzed. Moreover, the expression levels of FEZF1-AS1 and miR-196a in OSCC cells were detected via qRT-PCR. FEZF1-AS1 knockdown and miR-196a over-expression models were established using lentivirus transfection in OSCC cell lines (CAL-27 and Tca8113). Subsequently, the influences of FEZF1-AS1 and miR-196a on the biological functions of OSCC cells were analyzed via Cell Counting Kit-8 (CCK-8) assay, colony formation assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay, respectively. Furthermore, the potential mechanism was explored using the Luciferase reporter gene and recovery assays. The results of qRT-PCR proved that the expression level of FEZF1-AS1 in OSCC tissues was significantly higher than that of para-carcinoma tissues, and the difference was statistically significant. The pathological stage was significantly higher in patients with high-expression FEZF1-AS1 than those with low-expression FEZF1-AS1, while the overall survival rate was remarkably lower. The proliferation ability of cells in FEZF1-AS1 silencing group declined significantly when compared with the NC group. Similarly, qRT-PCR results verified that the expression of miR-196a in OSCC cell lines and tissues was significantly reduced as well. Meanwhile, the miR-196a expression was negatively correlated with FEZF1-AS1. Subsequent Luciferase reporter gene assay confirmed that overexpression of miR-196a could markedly reduce the activity of Luciferase containing wild-type FEZF1-AS1 vector rather than decrease the activity of Luciferase containing mutant-type vector or empty vector. These findings further indicated that FEZF1-AS1 could be targeted by miR-196a through this binding site. In addition, recovery assay demonstrates that there was a mutual regulatory effect between FEZF1-AS1 and miR-196a, jointly affecting the malignant progression of OSCC. The expression of lncRNA FEZF1-AS1 was markedly up-regulated in OSCC, which was significantly correlated with pathological stage and poor prognosis of OSCC patients. Therefore, it was believed that FEZF1-AS1 might promote the malignant progression of OSCC by regulating miR-196a.

VEGF and KRAS are Potential Targets of miR-206 Modulation in Triple Negative Breast Cancer

Triple negative is a subtype of breast cancer characterized by lack of expression of hormone receptors (ER, PR and Her2/neu). Due to the limited treatment options, the search for novel treatment targets continues. The aim of this study was to assess the differential expression of miR-206, VEGF and KRAS in TNBC and non-TNBC tissues and cell lines and to evaluate the modulatory effect of miR-206 on the key oncogenic targets VEGF and KRAS. The expression of miR-206, VEGF and KRAS was quantified using real time PCR in both paraffin embedded breast cancer and adjacent tissues as well as in MDA-MB-231 and MCF-7 cell lines. Cell lines were transfected with different concentrations of miR-206 mimic and their viability were assessed using MTT assay. Our results indicated that miR-206 was significantly downregulated in cancerous compared to non-cancerous tissues with a more pronounced downregulation in TNBC than non-TNBC tissues. VEGF and KRAS were significantly upregulated in TNBC compared to non-TNBC and their expression was negatively correlated to miR-206 expression. Transfection of TNBC and non-TNBC cell lines with miR-206 mimic resulted in a dose dependent reduction in cell viability as well as a significant reduction in VEGF and KRAS expression. In conclusion, based on our combined human tissues and cell line-based investigations we can suggest that VEGF and KRAS may be potential targets for miR-206-mediated regulation and that their targeting by miR-206 can be a highly efficient therapeutic strategy in TNBC.

Restoration of microRNA-29c in type I endometrioid cancer reduced endometrial cancer cell growth.

Endometrial cancer is the most common gynaecological cancer worldwide, and the prognosis of patients with advanced disease remains poor. MicroRNAs (miRs) are dysregulated in endometrial cancer. miRs-29-a, -b and -c expression levels are downregulated in endometrial cancer; however, a specific role for miR-29c and its target genes remain to be elucidated. The aim of the present study was to determine the functional effect of restoring miR-29c expression in endometrial cancer cell lines and to identify miR-29c targets involved in cancer progression. miR-29c expression in human endometrial tumour grades 1-3 and benign tissue as well as in the endometrial cancer cell lines Ishikawa, HEC1A and AN3CA were analysed using reverse transcriptase-quantitative PCR (RT-qPCR). The cell lines were transfected with miR-29c mimic, miR-29c inhibitor or scrambled control. xCELLigence real-time cell monitoring analysed proliferation and migration, and flow cytometry was used to analyse apoptosis and cell cycle. The expression of miR-29c target genes in transfected cell lines was analysed using RT-qPCR. miR-29c was downregulated in grade 1-3 endometrial cancer samples compared with benign endometrium. miR-29c was reduced in Ishikawa and AN3CA cells, but not in HEC1A cell lines compared with non-cancerous primary human endometrial epithelial cells. Overexpression of miR-29c variably reduced proliferation, increased apoptosis and reduced the expression levels of miR-29c target genes, including cell division cycle 42, HMG-box transcription factor 1, integrin subunit β 1, MCL1 apoptosis regulator BCL2 family member, MDM2 proto-oncogene, serum/glucocorticoid regulated kinase 1, sirtuin 1 and vascular endothelial growth factor A, across the three cell lines investigated. Inhibition of miR-29c in HEC1A cells increased proliferation and collagen type IV α 1 chain expression. The re-introduction of miR-29c to endometrial cancer cell lines reduced proliferation, increased apoptosis and reduced miR-29c target gene expression in vitro. The present results suggested that miR-29c may be a potential therapeutic target for endometrial cancer.

New insights into the roles of the FOXO3 and P27Kip1 genes in signaling pathways

Background: The forkhead box O3 (FOXO3) and p27Kip1 are two important genes in breast cancer progression. In the present study we analyzed the effect of simultaneous FOXO3 silencing and p27Kip1 activation on breast cancer cell survival and the potential targets of these changes in cancer molecular pathways.Materials and methods: The present study involved the cloning of FOXO3a shRNA and p27Kip1 genes under the control of the bidirectional survivin promoter to down- and up-regulate FOXO3 and p27Kip1 genes, respectively. After transfection of the recombinant expression vector into the breast cancer cell line, the inhibition of cell growth was assessed by MTS and flow cytometry assays. Following the extraction of total mRNA and protein, the expression of target genes was evaluated by qPCR and Western blotting in both treated and untreated cell lines. Then, the downstream protein responses were examined by 2 D electrophoresis. The differentially expressed proteins were also identified by mass spectrometry.Results: Rates of cell proliferation were significantly inhibited in the transfected cell line 72 h post-transfection. Proteomic profiling of the cell line resulted in the identification of seven novel protein markers in breast cancer responsive to these changes in expression of FOXO3 and p27Kip1. The changes in expression of these markers suggested that certain signaling pathways contribute to the development of breast cancer.Conclusion: Simultaneous silencing of FOXO3 and activation of p27Kip1 in MDA-MB-231 cells caused alterations in the expression level of several genes involved in apoptosis, cell proliferation, cell cycle control, tissue invasion, drug resistance, and metastasis. It seems that the identified genes might serve as useful biomarkers for breast cancer.

Abstract 2014: A novel small molecule inhibitor to suppress Notch1 activation in CRC

Abstract Introduction: Earlier we reported that aberrant overexpression of Notch1 in colon cancer patients is responsible for aggressiveness and progression of the disease. Current, therapeutic interventions of notch1 inhibitors that binds catalytic unit of γ-secretase and disrupts the cleavage activity cause gastrointestinal toxicity in clinical trial. Therefore, our study aims to develop a small molecule inhibitor, which inhibits Notch1 signaling but binds outside of γ-secretase’s catalytic unit thereby substantially reducing associated toxicity attributes. Through structure-activity relationship (SAR) studies based on naturally occurring Withaferin A (WA) structure, we have recently identified a WA-difuroate analog ASR458 as one such small molecule. Methods: To analyze the effect of ASR490 on HCT116 and HCT116/Notch1 cells, we performed phenotypic, western blot and in vivo analysis. Results: Notch1 overexpressing CRC cells (HCT/Notch1) showed aggressive cell growth (42%) as compared to HCT116 cells, however, ASR490 treatment inhibited cell growth in both HCT116 (IC50: 600 nM) as well as HCT/Notch1 cells (IC50: 850 nM). ASR458 significantly inhibited Notch1 expression and its downstream events in both the transfected cell lines. As a result, we observed shutdown of pro-survival machinery and induction of pro-apoptotic events (Bax, caspases and PARP) resulting in apoptotic cell death in ASR456 treated HCT116 and Notch1/HCT116 cells. Since Notch1 is responsible for epithelial mesenchyme transition (EMT); we analyzed EMT drivers and readout invasion/migration assays. ASR458 treatment reversed the mesenchymal characteristics by restoring the function of E-cadherin, which resulted in the suppression of invasion and migration in readout assays of HCT116 transfectants. Our ongoing Xenotransplanted mice experiments may suggest whether ASR458 inhibits the growth of HCT116 and HCT116/Notch1 tumors. Conclusion: Restrained gastrointestinal toxicity of Notch1 targeting therapeutics is achievable with novel small molecules displaying a differential binding affinity to γ-secretase. ASR458 inhibits cell growth in Notch1 overexpressing cells and abrogates Notch1 induced EMT. Ongoing molecular docking and in vivo studies may confirm ASR458 binding sites in γ-secretase and validate our in vitro findings respectively. Citation Format: Chendil Damodaran, Ashish Tyagi, Balaji Chandrasekaran, Venkatesh Kolluru, Ibrahim Jojua, Srinivasa Ramisetti, Arun K. Sharma, Murali K. Ankem. A novel small molecule inhibitor to suppress Notch1 activation in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2014.

Abstract 4527: Patient-derived organoids and xenografts identify neratinib plus HER2 antibody drug conjugate as a synergistic drug combination for HER2 mutated, nonamplified metastatic breast cancer

Abstract HER2 activating mutations are a novel, druggable genomic alteration in metastatic breast cancer (MBC). These HER2 mutations are predominantly found in HER2 gene amplification negative, hormone receptor positive breast cancers. We have previously demonstrated that HER2 mutations can be potently inhibited by the second generation, irreversible pan-HER tyrosine kinase inhibitor, neratinib (Bose et al., Cancer Discovery 2013). Further, we performed a phase II clinical trial to treat HER2 mutated MBC and we found that neratinib monotherapy produced a clinical benefit rate of 31% and progression free survival (PFS) of 16 weeks in a heavily pre-treated patient population (Ma et al., Clin. Can. Res. 2017). A second clinical trial, the SUMMIT trial (Hyman et al., Nature 2018), similarly showed a response rate of 32% and median PFS of 3.5 months for neratinib monotherapy for HER2 mutated, metastatic breast cancer. The objective of the current study is to explore novel combination strategies to improve the efficacy of neratinib in HER2 mutated breast cancer. In order to accelerate progress on testing multiple drug combinations, we developed organoids from two patient-derived xenografts (PDX’s) of HER2 mutated, ER positive metastatic breast cancer from our institution. We found that the ex vivoculture of these patient-derived organoids provides a platform to rapidly perform drug screens and drug combination testing on a scale that cannot be matched by other existing experimental platforms for patient-derived samples. The drug sensitivity of these organoids cultured ex vivo recapitulates the data previously obtained with transfected cell lines and in vivo experiments using PDX’s. Further, multiple drug combinations can be tested on these organoids in just two weeks, which is much shorter than the four to six months required for the corresponding slow-growing ER positive, breast cancer PDX’s that they are derived from. Strong, single agent activity was seen with neratinib, the HER2 antibody drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1), and the chemotherapy drug vinorelbine. Therefore, we tested combinations of neratinib plus the HER2 ADC and neratinib plus vinorelbine on these patient derived organoids. Neratinib plus HER2 ADC showed a strong drug synergy in both HER2 mutated organoids, as judged by the Loewe model of drug synergy. Prior publications suggest that the mechanism of action of neratinib in this combination is by increasing HER2 ubiquitylation and endocytic degradation, which will increase the uptake of the ADC that binds to HER2. We are now performing 384 well drug screens with these HER2 mutated, ER positive metastatic breast cancer organoids, and the results of the screens will be shown in our presentation. Citation Format: Shunqiang Li, Highkin Maureen, Tina M. Primeau, Stephanie L. Pratt, Irmina Diala, Richard E. Cutler, Grace Mann, Alshad S. Lalani, Cynthia X. Ma, Ron Bose. Patient-derived organoids and xenografts identify neratinib plus HER2 antibody drug conjugate as a synergistic drug combination for HER2 mutated, nonamplified metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4527.

Abstract 1773: The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer

Abstract Immune checkpoint inhibitors (ICIs), specifically those which target the T-cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated substantial clinical benefit in patients with non-small cell lung cancer (NSCLC). Tumoral PD-L1 expression may be important for response to therapy, but the precise mechanism of regulation has yet to be fully elucidated. It is known that PD-L1 expression is elevated in cancers harboring mutations in the RAS family of genes, specifically KRAS. Mutations in the KRAS gene are found in up to 30% of NSCLC tumors, and there are no targeted treatment options available for this subset of patients. The tyrosine phosphatase, SHP-2, has been shown to be a critical regulator of the KRAS signaling cascade. Therefore, genes regulated by the RAS/MAPK signaling cascade, including PD-L1, may be modulated by SHP-2 inhibition. Thus, this study aims to investigate the impact of SHP-2 activity on PD-L1 expression in NSCLC. The allosteric inhibitor of SHP-2, SHP099, was used to ablate SHP-2 activity in KRAS-mutant NSCLC cell lines, H460, A549, H2122, and EGFR-mutant NSCLC cell line PC9. PD-L1 levels were quantified by western blot and flow cytometry analyses. PD-L1 mRNA was measured by quantitative real time PCR (qRT-PCR) analysis. SHP099 activity was confirmed by SHP-2, siRNA transfection in KRAS active cell lines. Inhibition of SHP-2 led to increased levels of PD-L1 expression in the KRAS-mutant cell lines, but not in the EGFR-mutant cells. Analysis of PD-L1 surface expression by flow cytometry confirmed that surface PD-L1 levels increased following exposure to SHP099. To determine whether SHP-2 controlled expression at the transcriptional level, mRNA quantification by qRT-PCR displayed increased levels of PD-L1 mRNA following SHP099 treatment. Together, these results suggest that SHP-2 regulates PD-L1 expression in KRAS-active NSCLC at the level of PD-L1 gene expression. It is presently unclear whether SHP-2 also has a role in controlling protein turnover. Using SHP-2 mutant constructs that mimic a constitutively active phosphatase, we hope to gain further insight into the roles of both SHP-2 catalytic and scaffolding functionality on PD-L1 expression. Citation Format: Keller Toral. The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1773.

Research on the establishment of a TPM3 monoclonal stable transfected PANC-1 cell line and the experiment of the EMT occurrence in human pancreatic cancer.

Background: Pancreatic cancer is one of the most aggressive human malignancies that is associated with early metastasis and chemoresistance. Tropomyosin (TPM) is an indispensable regulatory protein for muscle contraction, Abnormal expressions of TPM gene are closely related to the carcinogenesis and metastasis of malignant tumors.Purpose: In this experiment, a monoclonal stable transfected cell line was established by the knock-down of TMP3 expression in PANC-1 cells with the lentivirus method, and the impacts of the downregulated TPM3 gene expression on the EMT-related molecules and biological behaviors of PANC-1 cells were explored.Methods: Based on the TPM3 gene sequence, we designed the RNA interference sequence, constructed and screened out the recombinant plasmid segment TPM3-shRNA with the optimal silencing effect, and carried out lentivirus titer determination and packaging. The recombinant lentiviral interference vector LV-TPM3-shRNA was transfected into PANC-1 cells; the transfection efficiency was then evaluated to screen out the monoclonal stable transfected PANC-1 cell line with downregulated TPM3 expression. The qRT-PCR and Western blot were used to detect the changes in the gene- and protein-levels expressions of EMT-related transcription factors in the target cell line and to respectively test the variations of the invasion and proliferation capacities.Results: It is shown that the monoclonal stable transfected PANC-1 cell line with downregulated TPM3 expression was successfully established with the recombinant lentiviral vector. After knocking down the expression of TPM3 gene in PANC-1 cells, EMT occurred in the cells; the cell phenotype showed malignant transformation, and the in vitro biological behaviors of the cells (such as proliferation and invasion) were enhanced to different degrees.Conclusion: It is indicated that the TPM3 gene can be a potential target spot for the treatment of pancreatic cancer.

Abstract 2554: Branched chain amino acid transaminase 1 in claudin-low breast cancer

Abstract Breast cancer, the most commonly diagnosed cancer in women, can be classified into five distinct subtypes. One subtype, claudin-low breast cancer, accounts for approximately 7% of the breast cancer cases and these tumors are aggressive and highly metastatic. RNA sequencing of human claudin-low breast cancers by other groups and RNA sequencing of a murine claudin-low mammary tumor cell line by our group has revealed that Bcat1 is significantly up-regulated in this breast cancer subtype. Bcat1 regulates the metabolism of branched chain amino acids and has been linked to numerous pathologies including heart disease, diabetes, and cancer. Based on this data we hypothesized that the expression of Bcat1 in claudin-low mammary tumors is driving the aggressive and metastatic nature of this cancer subtype and disrupting Bcat1 will deter these features. Elevated expression of Bcat1 in the murine claudin-low cell line RJ423, compared to the murine luminal mammary tumor cell line RJ345, has been confirmed at the mRNA and protein level. Bcat1 has been transiently down-regulated ~70% in RJ423 cells using siRNA and this suppression of Bcat1, contrary to the anticipated result, showed no effect on proliferation based on phospho-histone H3 immunofluorescence. However, when the cell population doubling time was investigated, a significant difference was seen between the RJ423 cell line and the cell lines with suppressed Bcat1 expression. To elucidate these contradicting results, cell cycle analysis using Bromodeoxyuridine and 7-AAD through flow cytometry is currently underway. RJ423 stably expressing Bcat1 shRNA and RJ345 stably expressing a Bcat1 expression vector are currently being selected. Further in vitro and in vivo studies will utilize these stably transfected cell lines. This study will determine whether further investigation into the effects of Bcat1 on claudin-low human breast cancer is prudent and if Bcat1 may be used as a therapeutic target. Citation Format: Lisa C. Reynen, Robert Jones, Roger Moorehead. Branched chain amino acid transaminase 1 in claudin-low breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2554.

Functionalized liposomal nanoparticles for efficient gene delivery system to neuronal cell transfection

Liposome based delivery systems provide a promising strategy for treatment of neurodegenerative diseases. A rational design of brain-targeted liposomes can support the development of more efficient treatments with drugs and gene materials. Here, we characterized surface modified liposomes with transferrin (Tf) protein and penetratin (Pen), a cell-penetrating peptide, for efficient and targeted gene delivery to brain cells. PenTf-liposomes efficiently encapsulated plasmid DNA, protected them against enzymatic degradation and exhibited a sustained in vitro release kinetics. The formulation demonstrated low cytotoxicity and was non-hemolytic. Liposomes were internalized into cells mainly through energy-dependent pathways especially clathrin-mediated endocytosis. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using PenTf-liposomes compared to unmodified liposomes. The ability of these liposomes to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. Rationally designed bifunctional targeted-liposomes provide an efficient tool for improving the targetability and efficacy of synthesized delivery systems. This investigation of liposomal properties attempted to address cell differences, as well as, vector differences, in gene transfectability. The findings indicate that PenTf-liposomes can be a safe and non-invasive approach to transfect neuronal cells through multiple endocytosis pathways.

Let-7i inhibits proliferation and migration of bladder cancer cells by targeting HMGA1

BackgroundLet-7 is one of the earliest discovered microRNAs(miRNAs) and has been reported to be down-regulated in multiple malignant tumors. The effects and molecular mechanisms of let-7i in bladder cancer are still unclear. This study was to investigate the effects and potential mechanisms of let-7i on bladder cancer cells.MethodsTotal RNA was extracted from bladder cancer cell lines. The expression levels of let-7i and HMGA1 were examined by quantitative real-time PCR. Cell viability was detected using the CCK-8 and colony formation assays, while transwell and wound healing assays were used to evaluate migration ability. Luciferase reporter assay and western blot were used to confirm the target gene of let-7i.ResultsCompared with the SV-40 immortalized human uroepithelial cell line (SV-HUC-1), bladder cancer cell lines T24 and 5637 had low levels of let-7i expression, but high levels of high mobility group protein A1 (HMGA1) expression. Transfection of cell lines T24 and 5637 with let-7i mimic suppressed cell proliferation and migration. Luciferase reporter assay confirmed HMGA1 may be one of the target genes of let-7i-5p. Protein and mRNA expression of HMGA1 was significantly downregulated in let-7i mimic transfected cell lines T24 and 5637.ConclusionsUp-regulation of let-7i suppressed proliferation and migration of the human bladder cancer cell lines T24 and 5637 by targeting HMGA1. These findings suggest that let-7i might be considered as a novel therapeutic target for bladder cancer.

INTERLEUKIN 1 RECEPTOR ANTAGONIST (IL-1RA) BIOLOGY IN ORAL EPITHELIUM, ORAL DYSPLASIA AND ORAL SQUAMOUS CELL CARCINOMA

Objectives Knowledge of molecular biology of oral dysplasia (OD) and oral squamous cell carcinoma (OSCC) is essential in order to find novel biological markers that could serve as predictor markers for malignant transformation. IL-1 receptor antagonist (IL-1RA), IL-1 natural inhibitor, is encoded by the IL1RN gene and has been reported to be downregulated in head and neck squamous cell carcinoma, but the effects of its downregulation in OSCC and OD are largely unknown. Thus, the aim of this research was to study the role of IL-1RA in oral carcinogenesis and oral keratinocyte senescence. Findings IL1RN, specifically intracellular IL-1RA type 1 (icIL-1RA1), is constitutively expressed in normal oral epithelium, but is downregulated, both in vitro and in vivo, in OD and OSCC cell lines. We also found an upregulation of IL-1R1 (IL-1 agonist receptor) in OSCC and OD cell lines. Using confocal microscopy, we have found that both proteins, IL-1RA and IL-1R1, are able to localize inside the nucleus, which suggests a new possible ways of interactions of intra-nuclear IL-1α in oral keratinocytes. Transient transfection in OSCC and OD cell lines with a plasmid encoding for icIL-1RA1, showed no or limited effects on cell migration (by cell exclusion and transwell assay), cell proliferation (by EDU incorporation) and IL-6 and IL-8 secretion (by ELISA). Preliminary data suggests an increase of IL-1 alpha and a decrease of icIL1-RA mRNA expression as primary oral keratinocytes and mortal OD cells senesce. Conclusions IL1RN is downregulated in oral dysplasia and oral cancer. How this downregulation favours oral carcinogenesis it is not yet known, but might be related with the oral senescence program.