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Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.

Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher ScientificTM has recently commercialized the Applied BiosystemsTM QuantStudioTM 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

Background Chromosome microdissection is one of the most important techniques in molecular cytogenetic research. Cotton (Gossypium Linnaeus, 1753) is the main natural fiber crop in the world. The resistance gene analog (RGA) cloning after its single chromosome microdissection can greatly promote cotton genome research and breeding. Results Using the linker adaptor PCR (LA-PCR) with the primers of rice disease-resistance homologues, three nucleotide sequences PS016 (KU051681), PS054 (KU051682), and PS157 (KU051680) were obtained from the chromosome Ah01 of upland cotton (cv. TM-1). The Blast results showed that the three sequences are the nucleotide binding site-leucine rich repeat (NBS-LRR) type RGAs. Clustering results indicated that they are homologous to these published RGAs. Thus, the three RGAs can definitely be confirmed as NBS-LRR class of RGAs in upland cotton. Conclusions Using single chromosome microdissection technique, DNA libraries containing cotton RGAs were obtained. This technique can promote cotton gene cloning, marker development and even the improvement of cotton genome research and breeding.

Rare DNA sequence variants hold important clinical and biological information, but are chal-lenging for existing methods (e.g. PCR, NGS) to profile in an inexpensive, multiplexed, simple-to-implement, and sequence-general way. Here, we present Blocker Displacement Amplification (BDA), a temperature-robust PCR method that selectively amplifies all sequence variants within a roughly 20 nt window by 1000-fold over wildtype sequences, allowing easy detection and quantitation of hundreds of potentials variants originally at ≤0.1% allele frequency. BDA employs a rationally designed competitive hybridization reaction to achieve similar enrichment performance across anneal temperatures ranging from 56°C to 64°C. This temperature robustness facilitates multiplexed enrichment of many different variants across the genome, and furthermore enables the use of in-expensive and portable thermocycling instruments for rare DNA variant detection. To show the sequence generality of BDA, we demonstrated enrichment on 156 single-nucleotide variants (SNVs). BDA has been validated on multiple different PCR platforms, DNA polymerases, and sample types including clinical cell-free DNA samples collected from the blood plasma of lung cancer patients. BDA quantitation of mutation allele fraction is generally consistent with deep sequencing results.

INTRODUCTION Mouse zygotes and early blastomeres have totipotent cell fate potential, generating both embryonic and extraembryonic cell lineages during normal development. This totipotent potential is gradually restricted during development, with the first cell fate specification event being completed by the blastocyst stage. Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) exhibit a pluripotent potential similar to that of the epiblast in blastocysts, efficiently generating all embryonic cell types but rarely contributing to extraembryonic lineages in the placenta and yolk sac. Experimentally, ESCs and iPSCs can be induced into cells with expanded developmental potential, albeit with low efficiency. Such pluripotent stem cells are characterized by their bidirectional developmental potential (contributing to both embryonic and extraembryonic lineages) and their strong induction of the MuERV-L (MERVL) endogenous retroviruses (ERVs), both of which are features of totipotent two-cell (2C) blastomeres. The low efficiency in generating bipotential ESCs reflects the existence of multiple cellular and molecular impediments that restrict the pluripotent cell fate potential. RATIONALE We identified miR-34a microRNA (miRNA) as a potent regulator that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. miR-34a −/− pluripotent stem cells exhibit a bidirectional cell fate potential, generating both embryonic and extraembryonic cell lineages in multiple functional assays. Hence, the miR-34a −/− pluripotent stem cells provide a powerful experimental system to dissect the molecular mechanisms that restrict cell fate potential in ESCs and iPSCs. RESULTS miR-34a −/− ESCs and iPSCs exhibited an expanded cell fate potential, generating both embryonic and extraembryonic lineages in teratomas, embryoid bodies, and chimeric embryos. In particular, a single miR-34a −/− ESC injected into a recipient morula could yield progenies in both inner cell mass (ICM) and trophectoderm. Expression profiling studies comparing wild-type and miR-34a −/− pluripotent stem cells revealed a strong and specific induction of the MERVL ERVs, together with many MERVL-proximal genes, in miR-34a −/− ESCs and iPSCs. Whereas wild-type ESCs and iPSCs almost exclusively expressed Oct4, miR-34a −/− ESCs and iPSCs were heterogeneous, containing mutually exclusive populations with either Oct4 expression or MERVL induction. Because MERVL is a specific and highly expressed molecular marker for totipotent 2C blastomeres and for bipotential ESCs, we investigated the mechanism by which miR-34a regulates MERVL expression. We demonstrated that MERVL induction in miR-34a –deficient pluripotent stem cells is regulated at the transcriptional level, at least in part because of an increase of the transcription factor Gata2, a direct target of miR-34a . Knockdown of gata2 in miR-34a –deficient pluripotent stem cells phenocopied miR-34a overexpression, not only down-regulating the expression of MERVL but also abolishing their bipotential cell fate. Thus, miR-34a restricts cell fate potential and represses MERVL induction in pluripotent stem cells, at least in part through down-regulation of Gata2. CONCLUSION We have identified miR-34a as a noncoding RNA that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. The miR-34a / gata2 /MERVL axis plays an essential role in modulating the transition between pluripotent stem cells and bipotential stem cells in culture. Thus, an intricate network of protein-coding genes, noncoding RNAs, and endogenous retroviruses could act cooperatively to define cell fate plasticity and developmental potential in pluripotent stem cells.

It is widely accepted that somatic cells can be reprogrammed by a set of transcription factors to become embryonic stem cell-like: These reprogrammed cells, induced pluripotent stem cells (iPSCs), are nearly identical to embryonic stem cells (ESCs), because both have the capacity to self-renew and to form all cellular lineages of the body. Transcriptional differences between ESCs, iPSCs, and fibroblasts can be analyzed by quantitative PCR (qPCR) using TaqMan(®) Gene Expression assays, a widely used tool for rapid analysis of different cell types. In this chapter, we describe the OpenArray(®) platform which generates qPCR data from high-throughput instrumentation. We examined the gene signature profiles of ESCs, fibroblasts, and iPSCs with a TaqMan(®) OpenArray(®) Human Stem Cell Panel containing 631 TaqMan(®) Gene Expression assays that represent pathways involved in self-renewal, pluripotency, lineage patterning, transcriptional networks, stem cell differentiation, and development.

Osteopenia and periarticular bony erosion are consequences of chronic inflammatory autoimmune disease due to an imbalance of osteoclast activity relative to new bone formation. Osteoclasts, which are specialized as the only bone resorbing cell type, are differentiated from hematopoietic myeloid precursor cells. Inflammatory signals mediated by multiple types of immune cells and cytokines have significant influence over osteoclast differentiation and function through direct effects on osteoclast precursors and indirect effects via osteoblasts and other cells in the bony microenvironment including synovial cells, stromal cells, osteocytes and chondrocytes. Recent studies have demonstrated that osteoclasts themselves express a number of immune receptors and are regulated similarly to macrophages and dendritic cells, closely related cells in the innate immune system. Though we are only beginning to understand the roles of innate immune receptors in osteoclasts, some of these receptors have been shown to be critical regulators of differentiation and function of osteoclasts. Osteoclasts likely function as the innate immune cells of the bone, thus are highly regulated to appropriately respond to stress and inflammatory changes in their microenvironment.

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86+ subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.

Objective Erosion of cartilage and bone is a hallmark of rheumatoid arthritis (RA). This study was undertaken to explore the roles of hyperproliferating synovial fibroblasts and macrophages in abnormal osteoclast formation, using the recently described BXD2 mouse model of RA. Methods Cell distribution in the joints was analyzed by immunohistochemistry, using tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclasts. To identify the defective cells in BXD2 mice, mouse synovial fibroblasts (MSFs) were cultured with bone marrow–derived macrophages. Osteoclast formation was assayed by TRAP staining and bone resorption pit assay, and the cytokine profiles of the MSFs and macrophages were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results In BXD2 mice, TRAP-positive osteoclasts were found at sites of active bone erosion, in close proximity to hyperproliferating synovial fibroblasts. On coculture, MSFs from BXD2 mice, but not C57BL/6 mice, produced high levels of RANKL messenger RNA, induced macrophages to form osteoclasts, and actively eroded bone slices, through a mechanism(s) that could be blocked by pretreatment with osteoprotegerin. Although macrophages from BXD2 mice expressed higher basal levels of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and IL-6 than those from C57BL/6 mice, abnormal osteoclast formation was not due to enhanced sensitivity of the BXD2 mouse macrophages to RANKL. TNFα, produced by both BXD2 MSFs and BXD2 mouse macrophages, had a strong stimulatory effect on RANKL expression. Conclusion BXD2 MSFs produce RANKL and induce the development of osteoclasts from macrophages. The enhanced production of RANKL is possibly due to autocrine stimulation, together with paracrine stimulation by factors produced by macrophages.

Antiestrogen compounds exhibit a variety of different effects in different tissues and are widely used for the treatment of osteoporosis, breast cancer, and other diseases. Upon examining the molecular mechanisms, we found that Smad4, a common signal transducer in the bone morphogenetic protein (BMP)/transforming growth factor-beta (TGF-beta) signaling pathway, functions as a transcription corepressor for human estrogen receptor alpha (ERalpha). Endogenous ERalpha was co-immunoprecipitated with Smad4, and the interaction was induced by antiestrogen ligands such as tamoxifen, raloxifene, and droloxifen, which was confirmed in chromatin immunoprecipitation assays. Smad4 and ERalpha form a complex when ERalpha binds to the estrogen-responsive element within the estrogen target gene promoter. Importantly, the expression of Smad4 inhibits both antiestrogen-induced luciferase activity and estrogen downstream target gene transcription in breast cancer cells. Mapping of the interaction domains indicates that the activation function 1 (AF1) domain of ERalpha is essential for its interaction with Smad4, while the MH1 domain and linker region of Smad4 are essential for the interaction. Our findings represent a novel mechanism that TGF-beta may regulate cell fate through Smad4-mediated cross-talk with estrogen.

Tumor suppressor Smad4 is the common signaling effector in the transforming growth factor beta (TGF-beta) superfamily. Phosphorylated regulatory Smads (R-Smads) interact with Smad4, and the complex translocates into the nucleus to regulate gene transcription. Proper TGF-beta signaling requires precise control of Smad functions. Smurfs have been shown to mediate the degradation of R-Smads but not the common-partner Smad4. We report a novel mechanism of Smad4 degradation. Jab1 interacts directly with Smad4 and induces its ubiquitylation for degradation. Jab1 was initially identified as a co-activator of c-Jun, and it also induces degradation of cell cycle inhibitor p27 and tumor suppressor p53. Ectopic expression of Jab1 decreased endogenous Smad4 steady-state levels. The 26S proteasome inhibitors lactacystin and MG132 reduced the degradation rate of Smad4 protein. Examination of the effects of JAB1-induced Smad4 degradation indicates that Jab1 inhibited TGF-beta-induced gene transcription. Our data suggest that Jab1 antagonizes TGF-beta function by inducing degradation of Smad4 through a distinct degradation pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that function to regulate gene expression at transcriptional and post-transcriptional level. Mature miRNAs are short, usually 18-23 nucleotides (nt) in length, with high homology in the seed region (position 2-8 from the 5′ end of a mature miRNA) within each family. This presents a great challenge when designing assays for quantitation by real-time polymerase chain reaction (PCR). Existing miRNA assays often trade in sensitivity and/or assay performance for specificity. Comparison between two different mature miRNAs with quantitative real-time PCR (qPCR) requires generating standard curves and characterizing PCR efficiency and sensitivity. Here we report a chip-based digital PCR technology. With this technology, we are able to accurately quantitate mature miRNAs without characterizing and optimizing the assay. Briefly, we input the same amount (ratio=1:1) of synthetic hsa-miR-19b and hsa-miR-92a. We then quantitated the miRNAs using reverse transcription followed by quantitative RT-PCR or chip-based QuantStudio™ 3D digital PCR (dPCR). Data from qPCR showed a delta Ct=2.58 between the cDNAs. This converted to a 3.84 fold difference between the two mature miRNAs, which deviated 284% from the input ratio, which equals 1. However, the data from dPCR indicated that the ratio between the amounts of the two mature miRNAs was 0.94 ± 0.02 (SD). Although statistically different, the results from dPCR deviated only 5% from the input ratio. Compared to qPCR, dPCR significantly enhanced the precision in sample quantitation. In summary, our study demonstrated that the chip-based dPCR technology has greatly increased precision in quantitating miRNA samples. The simplified workflow and improved output of dPCR are very useful features in broad applications such as revealing small changes in the miRNA expression levels. Citation Format: Yalei Wu, Victoria Dahlhoff, Lin He, Caifu Chen. Accurate quantitation of micro RNA by chip-based digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5241. doi:10.1158/1538-7445.AM2014-5241

[OBJECTIVES] Genetic alternations of EGFR and KRAS are frequently found in lung cancer cells. Accurate detection of these mutations from lung cancer peripheral blood may provide a noninvasive means for early cancer detection and disease monitoring. The goal of this study is to develop a quantitative analysis of EGFR and KRAS mutations both of ctDNA in plasma and of CTCs in blood cells from lung cancers by digital castPCR (competitive allele specific TaqMan-base PCR) technology. [METHODS] Molecular analysis and enumeration of CTCs in peripheral blood cells were analyzed by combining sample partition and digital castRT-qPCR assays without prior biophysical processing. The castPCR can detect rare copies of mutant alleles with a 6-log dynamic range and < 5-copy detection sensitivity for EGFR and KRAS mutations. Quantitative analysis of EGFR and KRAS mutations in ctDNA was done in OpenArray platform by digital castPCR. For CTC detection, whole blood samples with spiked-in known mutation and gen marker lung cancer cell lines were partitioned onto 96- or 384-well plate(s), such that each well contains either one cancer cell or none together with normal white blood cells and red blood cells. RNAs and/or DNAs were extracted by magnetic beads and were pre-amplified prior to molecular detection. Genetic mutations and cell type specific markers (CK19) for CTC identification and enumeration were determined by castRT-qPCR and TaqMan Gene expression (GEx) assays, respectively. Blood samples from lung cancer patients were first separated into blood cells and plasma portions before CTC detections. The sample plasma portions were used to detect EGFR and KRAS mutations of ctDNA by digital castPCR in OpenArray platform. [RESULTS] The sample partition process resulted in a relative CTC enrichment or digital enrichment of 20 - 400 folds (the relative ratio of CTC to normal cells) in a CTC-positive well. The RT-castPCR and TaqMan GEx assays accurately quantify the number of spiked-in tumor cells (10 - 60 cells/mL) in whole blood with known EGFR and/or KRAS mutations and CK19. For 10 blood samples from lung cancer patients, CTCs were detected from all patients even with stage I and II of 3 patients. In the plasma portions of the same samples, digital castPCR by OpenArray was able to detect EGFR mutations (p.L858R) and 19 mutations of EFGR exon 9. The results of EGFR mutations are consistent between intact CTCs and ctDNA. [CONCLUSION] Our preliminary data suggest that combination of digital sample enrichment and castRT-qPCR can be used to directly enumerate CTCs and detect cancer-related mutations in blood cells without prior biophysical sample enrichment. Digital castPCR by OpenArray can be used to assess circulating tumor DNA. Further test in larger clinical samples are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1736. doi:1538-7445.AM2012-1736