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Lung cancer is the world’s leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts. Genomic and transcriptomic analysis of lung adenocarcinoma (LUAD) in Asia indicates that Asian LUADs have fewer mutations, lower driver prevalence and fewer copy number alterations than European LUADs.

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types. National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work is supported in part by the Singapore National Medical Research Council grants (TCR/015-NCC/2016, CIRG18may-0057l, NMRC/CSA-SI/0018/2017, and NMRC/ OFIRG/0064/2017) and the National Research Foundation, Singapore (NRF-NRFF2015-04). W.Z. is supported in part by the National Key R&D Program of China (2018YFC1406902 and 2018YFC0910400), the National Natural Science Foundation of China (31970566), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDPB17). H.Y. is supported by the National Natural Science Foundation of China (32000407).

The human genome contains more than 200,000 gene isoforms. However, different isoforms can be highly similar, and with an average length of 1.5kb remain difficult to study with short read sequencing. To systematically evaluate the ability to study the transcriptome at a resolution of individual isoforms we profiled 5 human cell lines with short read cDNA sequencing and Nanopore long read direct RNA, amplification-free direct cDNA, PCR-cDNA sequencing. The long read protocols showed a high level of consistency, with amplification-free RNA and cDNA sequencing being most similar. While short and long reads generated comparable gene expression estimates, they differed substantially for individual isoforms. We find that increased read length improves read-to-transcript assignment, identifies interactions between alternative promoters and splicing, enables the discovery of novel transcripts from repetitive regions, facilitates the quantification of full-length fusion isoforms and enables the simultaneous profiling of m6A RNA modifications when RNA is sequenced directly. Our study demonstrates the advantage of long read RNA sequencing and provides a comprehensive resource that will enable the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution.

Simple Summary Metabolic adaptation by cancer cells is enabled through the rewiring of metabolic processes, thereby allowing them to survive and thrive in diverse tissue microenvironments. It is also exploited to maintain cancer stemness, drive epithelial–mesenchymal transition, and gain therapy resistance. These critical cellular events are pertinent to the various steps of cancer progression. Mechanistic insights into nutrient addiction arising from such metabolic rewiring have revealed therapeutic vulnerabilities that can be exploited as novel treatment modalities or for drug development. This review discusses concepts and principles of metabolic plasticity and highlights current preclinical and clinical strategies aimed at targeting these metabolic derangements. Abstract Cancer metabolism is a hallmark of cancer. Metabolic plasticity defines the ability of cancer cells to reprogram a plethora of metabolic pathways to meet unique energetic needs during the various steps of disease progression. Cell state transitions are phenotypic adaptations which confer distinct advantages that help cancer cells overcome progression hurdles, that include tumor initiation, expansive growth, resistance to therapy, metastasis, colonization, and relapse. It is increasingly appreciated that cancer cells need to appropriately reprogram their cellular metabolism in a timely manner to support the changes associated with new phenotypic cell states. We discuss metabolic alterations that may be adopted by cancer cells in relation to the maintenance of cancer stemness, activation of the epithelial–mesenchymal transition program for facilitating metastasis, and the acquisition of drug resistance. While such metabolic plasticity is harnessed by cancer cells for survival, their dependence and addiction towards certain metabolic pathways also present therapeutic opportunities that may be exploited.

The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune-ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune-ITH is associated with tumour transcriptomic-ITH, mutational burden and distinct immune microenvironments. Tumours with low immune-ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks contributing to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression., Intratumoural heterogeneity is a feature of liver cancer. Here, the authors demonstrate that heterogeneity exists at the immune cell level in liver cancer and show that tumours with high intratumoural immune heterogeneity demonstrated an immune suppressive microenvironment, which was associated with tumour evolution and a poor prognosis.

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson&rsquo, s disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/&beta, catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson&rsquo, s disease.

Here, we introduce UniPath, for representing single-cells using pathway and gene-set enrichment scores by a transformation of their open-chromatin or gene-expression profiles. Besides being robust to variability in dropout, UniPath provides consistency and scalability in estimating gene-set enrichment scores for every cell. UniPath’s approach of predicting temporal-order of single-cells using their gene-set activity score enables suppression of known covariates. UniPath based analysis of mouse cell atlas yielded surprising, albeit biologically-meaningful co-clustering of cell-types from distant organs and helped in annotating many unlabeled cells. By enabling unconventional analysis, UniPath also proves to be useful in inferring context-specific regulation in cancer cells.

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates., EGFR mutant lung adenocarcinoma (LUAD) exhibit diverse clinical outcomes in response to targeted therapies. Here the authors show that these LUADs involve a complex genomic landscape with high intratumor heterogeneity, providing insights into the evolutionary trajectory of oncogene-driven LUAD and potential mediators of EGFR TKI resistance.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

Background & Aims Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms. Methods We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured. Results Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non–small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation–related metabolites to generate ATP. Conclusions In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

Purpose Mesenchymal epithelial transition factor ( MET) activation has been implicated as an oncogenic driver in epidermal growth factor receptor ( EGFR)–mutant non–small-cell lung cancer (NSCLC) and can mediate primary and secondary resistance to EGFR tyrosine kinase inhibitors (TKI). High copy number thresholds have been suggested to enrich for response to MET inhibitors. We examined the clinical relevance of MET copy number gain (CNG) in the setting of treatment-naive metastatic EGFR-mutant–positive NSCLC. Patients and Methods MET fluorescence in situ hybridization was performed in 200 consecutive patients identified as metastatic treatment-naïve EGFR-mutant–positive. We defined MET-high as CNG greater than or equal to 5, with an additional criterion of MET/centromeric portion of chromosome 7 ratiο greater than or equal to 2 for amplification. Time-to-treatment failure (TTF) to EGFR TKI in patients identified as MET-high and -low was estimated by Kaplan-Meier method and compared using log-rank test. Multiregion single-nucleotide polymorphism array analysis was performed on 13 early-stage resected EGFR-mutant–positive NSCLC across 59 sectors to investigate intratumoral heterogeneity of MET CNG. Results Fifty-two (26%) of 200 patients in the metastatic cohort were MET-high at diagnosis; 46 (23%) had polysomy and six (3%) had amplification. Median TTF was 12.2 months (95% CI, 5.7 to 22.6 months) versus 13.1 months (95% CI, 10.6 to 15.0 months) for MET-high and -low, respectively ( P = .566), with no significant difference in response rate regardless of copy number thresholds. Loss of MET was observed in three of six patients identified as MET-high who underwent postprogression biopsies, which is consistent with marked intratumoral heterogeneity in MET CNG observed in early-stage tumors. Suboptimal response (TTF, 1.0 to 6.4 months) to EGFR TKI was observed in patients with coexisting MET amplification (five [3.2%] of 154). Conclusion Although up to 26% of TKI-naïve EGFR-mutant–positive NSCLC harbor high MET CNG by fluorescence in situ hybridization, this did not significantly affect response to TKI, except in patients identified as MET-amplified. Our data underscore the limitations of adopting arbitrary copy number thresholds and the need for cross-assay validation to define therapeutically tractable MET pathway dysregulation in EGFR-mutant–positive NSCLC.

Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity, that fuel tumor growth and contribute to the heterogeneous nature of tumors. First identified in hematological malignancies, CSC populations have to date been proposed in solid tumors in various organs. In vitro and in vivo assays, mouse genetic models, and more recently single-cell sequencing technologies and other '-omics' methodologies have not only facilitated the identification of novel CSC populations but also revealed and clarified novel properties of CSCs. Increasingly, both cell-autonomous and CSC niche factors are recognized as important contributors of CSC properties. The deepened understanding of CSC properties and characteristics would enable and facilitate the rational design of CSC-specific therapeutics that would, ideally, have high selectivity for cancer cells, eliminate tumor bulk, and prevent tumor recurrence. Addressing these issues would form some of the key challenges of the CSC research field in the coming years.

Recent advances in single-cell open-chromatin and transcriptome profiling have created a challenge of exploring novel applications with a meaningful transformation of read-counts, which often have high variability in noise and drop-out among cells. Here, we introduce UniPath, for representing single-cells using pathway and gene-set enrichment scores by a transformation of their open-chromatin or gene-expression profiles. The robust statistical approach of UniPath provides high accuracy, consistency and scalability in estimating gene-set enrichment scores for every cell. Its framework provides an easy solution for handling variability in drop-out rate, which can sometimes create artefact due to systematic patterns. UniPath provides an alternative approach of dimension reduction of single-cell open-chromatin profiles. UniPath's approach of predicting temporal-order of single-cells using their pathway enrichment scores enables suppression of covariates to achieve correct order of cells. Analysis of mouse cell atlas using our approach yielded surprising, albeit biologically-meaningful co-clustering of cell-types from distant organs. By enabling an unconventional method of exploiting pathway co-occurrence to compare two groups of cells, our approach also proves to be useful in inferring context-specific regulations in cancer cells. Available at https://reggenlab.github.io/UniPathWeb/.

AXL is a receptor tyrosine kinase that is often overexpressed in many cancers. It contributes to tumor progression, metastasis and drug resistance through activating downstream signaling cascades, making it an emerging therapeutic target. Moreover, the first-in-class AXL inhibitor R428 (BGB321) was lately approved by the FDA for the treatment of relapsed or refractory acute myeloid leukemia. Previously, R428 (BGB321) was also reported to show selective sensitivity towards ovarian cancers with a Mesenchymal molecular subtype. Recently, a novel role of AXL in the regulation of DNA damage responses has been brought to light. Here, we further explored AXL's role in mediating DNA damage responses by using ovarian cancer as a disease model. AXL inhibition using R428 resulted in the increase DNA damage foci in ovarian cancer cells SKOV3 and HeyA8. This occurred concurrently with the upregulation of classic DNA damage response signaling molecules such as γH2Ax, ATM and ATR. RNA sequencing results further validated this observation, that R428-treated cells showed downregulation of genes involved in cell cycle control and DNA replication, suggesting the decrease in proliferation. Through SILAC co-immunoprecipitation mass spectrometry, we uncovered a novel binding partner of AXL that could potentially regulate the DNA damage repair upon the inhibition of AXL, giving us insights to how AXL inhibition might contribute to DNA damage. Furthermore, our results showed that AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress, paving ways to the rationale for potential combinatory use of AXL and DNA damage repair inhibitors. Citation Format: Xun Hui Yeo, Wai Leong Tam, Ruby Yun-Ju Huang. Role of receptor tyrosine kinase AXL in mediating DNA damage response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4198.

During cancer progression, cells would reversibly transit among different cell states that differ in their competence to contribute towards tumor growth, survival, and metastasis. The activation of epithelial-mesenchymal transition (EMT) program in cancer cells has often been attributed to enhanced tumorigenic potential and a gain of therapeutic resistance. We hypothesized that by promoting mesenchymal-epithelial transition (MET), the reverse process of EMT, it could induce a loss of tumorigenic potential and increase susceptibility to standard chemotherapeutics. As EMT is a dynamic and reversible process, epigenetic regulations such as histone modifications and DNA methylation are important in regulating and reprogramming the expression of EMT-responsive genes. Hence, we seek to understand how the epigenetic landscape is altered during transitions between cell states. To understand the molecular link between epigenetic regulations and cell state transitions in cancer cells, a high-throughput library screen on epigenetic modifiers was performed. Epigenetic modifying compounds that can induce MET in basal-like breast cancer cells were uncovered. In vitro and in vivo data demonstrate that compounds targeting the non-BET bromodomains were able to promote MET, induce a loss of stem-like properties, and decrease the tumorigenicity of basal-like breast cancer cells. Using various next-generation sequencing approaches, we also elucidated the mechanisms of how targeting non-BET bromodomains would regulate cell state transitions in breast cancers. While epigenetic therapies are effective in the treatment of hematological malignancies, its application in solid tumors is still limited. Here, we propose the possible use of an epigenetic MET inducer for differentiation therapy of the aggressive basal-like breast cancers. Citation Format: Li Ping Toh, Ser Yue Loo, Wai Leong Tam. Epigenetic regulation of cell state transitions and the therapeutic implications in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3833.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA.

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC-specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial-to-mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti-TIC activity with low toxicity to non-TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.

Tumour-initiating cells (TICs) are responsible for metastatic dissemination and clinical relapse in a variety of cancers. Analogies between TICs and normal tissue stem cells have led to the proposal that activation of the normal stem-cell program within a tissue serves as the major mechanism for generating TICs. Supporting this notion, we and others previously established that the Slug epithelial-to-mesenchymal transition-inducing transcription factor (EMT-TF), a member of the Snail family, serves as a master regulator of the gland-reconstituting activity of normal mammary stem cells, and that forced expression of Slug in collaboration with Sox9 in breast cancer cells can efficiently induce entrance into the TIC state. However, these earlier studies focused on xenograft models with cultured cell lines and involved ectopic expression of EMT-TFs, often at non-physiological levels. Using genetically engineered knock-in reporter mouse lines, here we show that normal gland-reconstituting mammary stem cells residing in the basal layer of the mammary epithelium and breast TICs originating in the luminal layer exploit the paralogous EMT-TFs Slug and Snail, respectively, which induce distinct EMT programs. Broadly, our findings suggest that the seemingly similar stem-cell programs operating in TICs and normal stem cells of the corresponding normal tissue are likely to differ significantly in their details.

Tumor recurrence remains the main reason for breast cancer-associated mortality, and there are unmet clinical demands for the discovery of new biomarkers and development of treatment solutions to benefit patients with breast cancer at high risk of recurrence. Here we report the identification of chromosomal copy-number amplification at 1q21.3 that is enriched in subpopulations of breast cancer cells bearing characteristics of tumor-initiating cells (TICs) and that strongly associates with breast cancer recurrence. Amplification is present in ∼10-30% of primary tumors but in more than 70% of recurrent tumors, regardless of breast cancer subtype. Detection of amplification in cell-free DNA (cfDNA) from blood is strongly associated with early relapse in patients with breast cancer and could also be used to track the emergence of tumor resistance to chemotherapy. We further show that 1q21.3-encoded S100 calcium-binding protein (S100A) family members, mainly S100A7, S100A8, and S100A9 (S100A7/8/9), and IL-1 receptor-associated kinase 1 (IRAK1) establish a reciprocal feedback loop driving tumorsphere growth. Notably, this functional circuitry can be disrupted by the small-molecule kinase inhibitor pacritinib, leading to preferential impairment of the growth of 1q21.3-amplified breast tumors. Our study uncovers the 1q21.3-directed S100A7/8/9-IRAK1 feedback loop as a crucial component of breast cancer recurrence, serving as both a trackable biomarker and an actionable therapeutic target for breast cancer.

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)--a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)--is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44(high)CD24(low) population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

The tumour-initiating cell (TIC) model accounts for phenotypic and functional heterogeneity among tumour cells. MicroRNAs (miRNAs) are regulatory molecules frequently aberrantly expressed in cancers, and may contribute towards tumour heterogeneity and TIC behaviour. More recent efforts have focused on miRNAs as diagnostic or therapeutic targets. Here, we identified the TIC-specific miRNAs, miR-1246 and miR-1290, as crucial drivers for tumour initiation and cancer progression in human non-small cell lung cancer. The loss of either miRNA impacted the tumour-initiating potential of TICs and their ability to metastasize. Longitudinal analyses of serum miR-1246 and miR-1290 levels across time correlate their circulating levels to the clinical response of lung cancer patients who were receiving ongoing anti-neoplastic therapies. Functionally, direct inhibition of either miRNA with locked nucleic acid administered systemically, can arrest the growth of established patient-derived xenograft tumours, thus indicating that these miRNAs are clinically useful as biomarkers for tracking disease progression and as therapeutic targets., miRNAs can function either as proto-oncogenes or tumour suppressors in several cancers; however their function in tumour initiating cells is unclear. Here, Zhang et al. show that tumour initiating cell-specific miR-1246 and miR-1290 promote lung cancer initiation and metastasis and could serve as prognostic markers.

Have cancer stem cells MET their match? Solid tumors have been hypothesized to contain a subset of highly aggressive cells that fuel tumor growth and metastasis. The search is on for drugs that selectively kill or diminish the malignant properties of these tumor-initiating cells (TICs; previously called “cancer stem cells”). Pattabiraman et al. hypothesized that compounds that induce TICs to undergo a phenotypic change called the mesenchymal-to-epithelial transition (MET) would therefore cause TICs to lose their tumor-initiating ability. Indeed, drugs activating the protein kinase A signaling pathway triggered an epigenetic reprogramming of TICs that resulted in the cells acquiring a more benign epithelial-like phenotype. Science , this issue p. 10.1126/science.aad3680

SummaryRegulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.

Metastatic tumour recurrence due to failed treatments remains a major challenge of breast cancer clinical management. Here we report that interleukin-1 receptor-associated kinase 1 (IRAK1) is overexpressed in a subset of breast cancers, in particular triple-negative breast cancer (TNBC), where it acts to drive aggressive growth, metastasis and acquired resistance to paclitaxel treatment. We show that IRAK1 overexpression confers TNBC growth advantage through NF-κB-related cytokine secretion and metastatic TNBC cells exhibit gain of IRAK1 dependency, resulting in high susceptibility to genetic and pharmacologic inhibition of IRAK1. Importantly, paclitaxel treatment induces strong IRAK1 phosphorylation, an increase in inflammatory cytokine expression, enrichment of cancer stem cells and acquired resistance to paclitaxel treatment. Pharmacologic inhibition of IRAK1 is able to reverse paclitaxel resistance by triggering massive apoptosis at least in part through inhibiting p38-MCL1 pro-survival pathway. Our study thus demonstrates IRAK1 as a promising therapeutic target for TNBC metastasis and paclitaxel resistance., Triple negative breast cancer (TNBC) patients often acquire resistant to chemotherapy. In this study, the authors identify the IRAK1 as the crucial driver of NF-κB-related cytokine secretion involved in TNBC metastasis and therapy resistance.

The transcription factor Sox2 is a master regulator that maintains stemness in embryonic stem cells and neural stem cells. Using elegant lineage tracing strategies and genetic reporter mouse models, two studies (one of which is by Vanner and colleagues in this issue of Cancer Cell) now demonstrate that rare Sox2-expressing cells are the founding cancer stem cell population driving tumor initiation and therapy resistance.

Hundreds of microRNAs (miRNAs) are expressed in mammalian cells, where they aid in modulating gene expression by mediating mRNA transcript cleavage and/or regulation of translation rate. Functional studies to date have demonstrated that several of these miRNAs are important during development. However, the role of miRNAs in the regulation of stem cell growth and differentiation is not well understood. We show herein that microRNA (miR)-134 levels are maximally elevated at day 4 after retinoic acid-induced differentiation or day 2 after N2B27-induced differentiation of mouse embryonic stem cells (mESCs), but this change is not observed during embryoid body differentiation. The elevation of miR-134 levels alone in mESCs enhances differentiation toward ectodermal lineages, an effect that is blocked by a miR-134 antagonist. The promotion of mESC differentiation by miR-134 is due, in part, to its direct translational attenuation of Nanog and LRH1, both of which are known positive regulators of Oct4/POU5F1 and mESC growth. Together, the data demonstrate that miR-134 alone can enhance the differentiation of mESCs to ectodermal lineages and establish a functional role for miR-134 in modulating mESC differentiation through its potential to target and regulate multiple mRNAs. Disclosure of potential conflicts of interest is found at the end of this article.

The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis.

Embryonic stem (ES) cells are pluripotent cells that can self-renew or differentiate into many cell types. A unique network of transcription factors and signalling molecules are essential for maintaining this capability. Here, we report that a spalt family member, Sall4, is required for the pluripotency of ES cells. Similarly to Oct4, a reduction in Sall4 levels in mouse ES cells results in respecification, under the appropriate culture conditions, of ES cells to the trophoblast lineage. Sall4 regulates transcription of Pou5f1 which encodes Oct4. Sall4 binds to the highly conserved regulatory region of the Pou5f1 distal enhancer and activates Pou5f1 expression in vivo and in vitro. Microinjection of Sall4 small interfering (si) RNA into mouse zygotes resulted in reduction of Sall4 and Oct4 mRNAs in preimplantation embryos and significant expansion of Cdx2 expression into the inner cell mass. These results demonstrate that Sall4 is a transcriptional activator of Pou5f1 and has a critical role in the maintenance of ES cell pluripotency by modulating Oct4 expression. The data also indicates that Sall4 is important for early embryonic cell-fate decisions.

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types., National Institutes of Health (U.S.) (Grant RO1-CA084198), National Institutes of Health (U.S.) (Grant U01-CA184897), National Institutes of Health (U.S.) (Grant R01-GM085319), National Science Foundation (U.S.). Graduate Research Fellowship (Grant 1122374)

Embryonic stem cells (ESCs) are pluripotent cells that can either self-renew or differentiate into many cell types. Oct4 and Sox2 are transcription factors essential to the pluripotent and self-renewing phenotypes of ESCs. Both factors are upstream in the hierarchy of the transcription regulatory network and are partners in regulating several ESC-specific genes. In ESCs, Sox2 is transcriptionally regulated by an enhancer containing a composite sox-oct element that Oct4 and Sox2 bind in a combinatorial interaction. It has previously been shown that Pou5f1, the Oct4 gene, contains a distal enhancer imparting specific expression in both ESCs and preimplantation embryos. Here, we identify a composite sox-oct element within this enhancer and show that it is involved in Pou5f1 transcriptional activity in ESCs. In vitro experiments with ESC nuclear extracts demonstrate that Oct4 and Sox2 interact specifically with this regulatory element. More importantly, by chromatin immunoprecipitation assay, we establish that both Oct4 and Sox2 bind directly to the composite sox-oct elements in both Pou5f1 and Sox2 in living mouse and human ESCs. Specific knockdown of either Oct4 or Sox2 by RNA interference leads to the reduction of both genes' enhancer activities and endogenous expression levels in addition to ESC differentiation. Our data uncover a positive and potentially self-reinforcing regulatory loop that maintains Pou5f1 and Sox2 expression via the Oct4/Sox2 complex in pluripotent cells.

The cell-biological program termed the epithelial-mesenchymal transition (EMT) confers on cancer cells mesenchymal traits and an ability to enter the cancer stem cell (CSC) state. However, the interactions between CSCs and their surrounding microenvironment are poorly understood. Here we show that tumour-associated monocytes and macrophages (TAMs) create a CSC niche through juxtacrine signalling with CSCs. We performed quantitative proteomic profiling and found that the EMT program upregulates the expression of CD90, also known as Thy1, and EphA4, which mediate the physical interactions of CSCs with TAMs by directly binding with their respective counter-receptors on these cells. In response, the EphA4 receptor on the carcinoma cells activates Src and NF-κ B. In turn, NF-κ B in the CSCs induces the secretion of a variety of cytokines that serve to sustain the stem cell state. Indeed, admixed macrophages enhance the CSC activities of carcinoma cells. These findings underscore the significance of TAMs as important components of the CSC niche., National Institutes of Health (U.S.) (Grant R01-CA078461), National Institutes of Health (U.S.) (Grant P01-CA080111), National Institutes of Health (U.S.) (Grant U54-CA163109)

Epithelial-mesenchymal transitions (EMTs) are a key requirement for cancer cells to metastasize and colonize in a new environment. Epithelial-mesenchymal plasticity is mediated by master transcription factors and is also subject to complex epigenetic regulation. This Review outlines our current understanding of the interactions between EMT-inducing transcription factors and epigenetic modulators during cancer progression and the therapeutic implications of exploiting this intricate regulatory process. During the course of malignant cancer progression, neoplastic cells undergo dynamic and reversible transitions between multiple phenotypic states, the extremes of which are defined by the expression of epithelial and mesenchymal phenotypes. This plasticity is enabled by underlying shifts in epigenetic regulation. A small cohort of pleiotropically acting transcription factors is widely recognized to effect these shifts by controlling the expression of a constituency of key target genes. These master regulators depend on complex epigenetic regulatory mechanisms, notably the induction of changes in the modifications of chromatin-associated histones, in order to achieve the widespread changes in gene expression observed during epithelial-mesenchymal transitions (EMTs). These associations indicate that an understanding of the functional interactions between such EMT-inducing transcription factors and the modulators of chromatin configuration will provide crucial insights into the fundamental mechanisms underlying cancer progression and may, in the longer term, generate new diagnostic and therapeutic modalities for treating high-grade malignancies.

The epithelial-mesenchymal transition program becomes activated during malignant progression and can enrich for cancer stem cells (CSCs). We report that inhibition of protein kinase C α (PKCα) specifically targets CSCs but has little effect on non-CSCs. The formation of CSCs from non-stem cells involves a shift from EGFR to PDGFR signaling and results in the PKCα-dependent activation of FRA1. We identified an AP-1 molecular switch in which c-FOS and FRA1 are preferentially utilized in non-CSCs and CSCs, respectively. PKCα and FRA1 expression is associated with the aggressive triple-negative breast cancers, and the depletion of FRA1 results in a mesenchymal-epithelial transition. Hence, identifying molecular features that shift between cell states can be exploited to target signaling components critical to CSCs., National Cancer Institute (U.S.) (Grant P01-CA080111), National Institutes of Health (U.S.) (Grant R01-CA078461)

The cell-biological program termed the epithelial-mesenchymal transition (EMT) confers on cancer cells mesenchymal traits and an ability to enter the cancer stem cell (CSC) state. In addition, EMT upregulates the expression of cell surface receptors that enable CSCs to interact with their surrounding microenvironment in order to sustain their stem cell state. We discovered that EphA4 and CD90, two cell surface proteins upregulated in the carcinoma cells by the EMT, mediate juxtacrine signalling between the CSCs and tumor-associated monocytes and macrophages (TAMs). When engaged with its counter-receptor on TAMs, EphA4 on the CSCs activates Src and PLCγ1, the latter resulting in the translocation of NF-κB into the nucleus, which cooperates with Twist to drive the rapid induction of a variety of cytokines in the CSCs. Among the secreted cytokines IL-8 and IL-6 maintain the CSC-state by reinforcing the expression of the EMT-inducing transcription factors, whereas other cytokines GM-CSF, M-CSF and IL-10 recruit monocytes and induce their differentiation into M2-like macrophages. Together these cytokines perpetuate the CSC-promoting interactions between CSCs and TAMs. Indeed, admixed TAMs promote the tumor-initiating ability of CSCs whereas ablation of endogenous macrophages substantially diminished tumor initiation. Importantly, the juxtaposition of TAMs with the mesenchymal-like CD90high CSCs can be observed at the invading edge in primary human breast tumor sections and xenograft tumors. On the contrary, we rarely find TAMS infiltrating beyond the edge of tumors into the regions where the CD90neg non-stem carcinoma cells reside. These findings underscore the importance of TAMs as critical components of the CSC niche and highlight their potential as therapeutics targets. Citation Format: Haihui Lu, Wai Leong Tam, Vera Donnenberg, Xin Ye, Rohit Bhargava, Robert Weinberg. Juxtacrine signaling between the tumor-associated macrophages and the breast cancer stem cells contribute to the cancer stem cell niche by inducing a cytokine signaling network. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B150.

Publisher Summary This chapter provides insights into the interface between transcription and translational networks, and discusses some lessons gained from deciphering the reciprocal interplay between the different regulatory mechanisms. Transcription factors are important determinants of cellular identity where their expression can shift the balance of cell fate. A recent class of small non-coding RNAs discovered, microRNAs (miRNAs), also appears to have determining roles in the control of cellular phenotypes, development, and disease. Chromatin immunoprecipitation (ChIP) is a technique used in identifying the DNA binding sites of transcription factors in the cell. The seven or eight nucleotides at the 5' end of a miRNA that has been postulated to serve as the primary determinant of target specificity. The interface between miRNAs and transcription factor networks presents a new dimension toward the comprehensive understanding of the molecular architecture of the cell. The miRNA gene signatures in diseases that affect the cardiovascular, nervous, and immune systems have been reported. The use of miRNAs as anti-tumorigenic agents in cancer and as re-programming factors for generating stem cells highlights the value of miRNAs as therapeutic agents. The major advantage is that these small molecules can be chemically synthesized and modified to have long half life. They may also be easier to administer owing to their size and stability. Distinction between real physiological interactions from non-specific ones resulting from miRNA misexpression is required to understand the rules that govern miRNA targeting of transcription regulators. This in turn will be valuable in overcoming off-target effects.

Aggressive forms of cancer are often defined by recurrent chromosomal alterations, yet in most cases, the causal or contributing genetic components remain poorly understood. Here, we utilized microarray informatics to identify candidate oncogenes potentially contributing to aggressive breast cancer behavior. We identified the Rab-coupling protein RCP (also known as RAB11FIP1), which is located at a chromosomal region frequently amplified in breast cancer (8p11-12) as a potential candidate. Overexpression of RCP in MCF10A normal human mammary epithelial cells resulted in acquisition of tumorigenic properties such as loss of contact inhibition, growth-factor independence, and anchorage-independent growth. Conversely, knockdown of RCP in human breast cancer cell lines inhibited colony formation, invasion, and migration in vitro and markedly reduced tumor formation and metastasis in mouse xenograft models. Overexpression of RCP enhanced ERK phosphorylation and increased Ras activation in vitro. As these results indicate that RCP is a multifunctional gene frequently amplified in breast cancer that encodes a protein with Ras-activating function, we suggest it has potential importance as a therapeutic target. Furthermore, these studies provide new insight into the emerging role of the Rab family of small G proteins and their interacting partners in carcinogenesis.

While much attention has been given to the study of different genetic and chemical methods for the generation of iPS (induced pluripotent stem) cell lines, relatively little is known about the variability in overall quality of iPS cells. This paper identifies a transcription factor, Tbx3, that significantly improves the quality of iPS cells. Tbx3 also accelerates the reprogramming process of mouse embryonic fibroblasts into iPS cells and significantly improves the germ-line transmission of iPS-derived germ cells in chimaeric animals. The transcription factor Tbx3 is shown to significantly improve the quality of induced pluripotent stem (iPS) cells. Tbx3 binding sites in embryonic stem cells are present in genes involved in pluripotency and reprogramming factors. Furthermore, there are intrinsic qualitative differences in iPS cells generated by different methods in terms of their pluripotency, thus highlighting the need to rigorously characterize iPS cells beyond in vitro studies. Induced pluripotent stem (iPS) cells can be obtained by the introduction of defined factors into somatic cells1. The combination of Oct4 (also known as Pou5f1), Sox2 and Klf4 (which we term OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts. These cells are thought to resemble embryonic stem cells (ESCs) on the basis of global gene expression analyses; however, few studies have tested the ability and efficiency of iPS cells to contribute to chimaerism, colonization of germ tissues, and most importantly, germ-line transmission and live birth from iPS cells produced by tetraploid complementation. Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.

The Wnt signaling pathway is necessary both for maintaining undifferentiated stem cells and for directing their differentiation. In mouse embryonic stem cells (ESCs), Wnt signaling preferentially maintains “stemness” under certain permissive conditions. T-cell factor 3 (Tcf3) is a component of the Wnt signaling and a dominant downstream effector in ESCs. Despite the wealth of knowledge regarding the importance of Wnt signaling underlying stem cells functions, the precise mechanistic explanation by which the effects are mediated is unknown. In this study, we identified new regulatory targets of Tcf3 using a whole-genome approach and found that Tcf3 transcriptionally represses many genes important for maintaining pluripotency and self-renewal, as well as those involved in lineage commitment and stem cell differentiation. This effect is in part mediated by the corepressors transducin-like enhancer of split 2 and C-terminal Binding Protein (CtBP). Notably, Tcf3 binds to and represses the Oct4 promoter, and this repressive effect requires both the Groucho and CtBP interacting domains of Tcf3. Interestingly, we find that in mouse preimplantation development embryos, Tcf3 expression is coregulated with Oct4 and Nanog and becomes localized to the inner cell mass of the blastocyst. These data demonstrate an important role for Tcf3 in modulating the appropriate level of gene transcription in ESCs and during embryonic development. Disclosure of potential conflicts of interest is found at the end of this article.

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death., published_or_final_version

Stem cells are biologically and clinically important cells characterized by their ability to self-renew or differentiate into many cell types. The decision between self-renewal and differentiation is governed by extracellular signals, coupled to intracellular signaling cascades which activate transcription programs. Hence, transcription regulation plays a determining role in conferring cellular identity and function. As intrinsic determinants, transcription factors provide an entry point for uncovering how stem cells attain their phenotype, and how lineage-specific differentiation is initiated. The step-wise maturation of stem cells into terminally differentiated cell types requires the timely activation of a cascade of transcription programs governed by lineage-specifying transcription factors. The identification of key regulators in the hierarchy and their targets can provide clues into the manipulation of stem cells. This review examines the role of key transcription factors in various stem cell types, and emphasizes on their control of downstream gene expression, which ultimately contributes to cellular function.

Ageing is often defined in the context of telomerase activity and telomere length regulation. Most somatic cells have limited replication ability and undergo senescence eventually. Stem cells are unique as they possess more abundant telomerase activity and are able to maintain telomere lengths for a longer period. Embryonic stem cells are particularly resistant to ageing and can be propagated indefinitely. Remarkably, adult somatic cells can be reprogrammed to an ESC-like state by various means including cell fusion, exposure to ESC cell-free extracts, enforced expression of specific molecules, and somatic cell nuclear transfer. Thus, the rejuvenation of an 'aged' state can be effected by the activation of specific key molecules in the cell. Here, we argue that cellular ageing is a reversible process, and this is determined by the balance of biological molecules which directly or indirectly control telomere length and telomerase activity, either through altering gene expression and/or modulating the epigenetic state of the chromatin.

SummaryWe present rna22, a method for identifying microRNA binding sites and their corresponding heteroduplexes. Rna22 does not rely upon cross-species conservation, is resilient to noise, and, unlike previous methods, it first finds putative microRNA binding sites in the sequence of interest, then identifies the targeting microRNA. Computationally, we show that rna22 identifies most of the currently known heteroduplexes. Experimentally, with luciferase assays, we demonstrate average repressions of 30% or more for 168 of 226 tested targets. The analysis suggests that some microRNAs may have as many as a few thousand targets, and that between 74% and 92% of the gene transcripts in four model genomes are likely under microRNA control through their untranslated and amino acid coding regions. We also extended the method's key idea to a low-error microRNA-precursor-discovery scheme; our studies suggest that the number of microRNA precursors in mammalian genomes likely ranges in the tens of thousands.

It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the “mesenchymal metabolic signature” (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits., United States. National Institutes of Health (RO1 CA103866), United States. National Institutes of Health (AI047389), United States. National Institutes of Health (K99 CA168940), American Cancer Society (PF-12-099-01-TGB), American Cancer Society (PF-13-356-01-TBE), United States. Department of Defense (BC123066), United States. National Institutes of Health (CA112967), United States. National Institutes of Health (ES015339)