Estrogen regulation and functional role of FGFR4 in estrogen receptor positive breast cancer.

Background: Resistance to endocrine therapy is a major challenge of managing estrogen receptor positive (ER+) breast cancer. We previously reported frequent overexpression of FGFR4 in endocrine resistant cell lines and breast cancers that recurred and metastasized following endocrine therapy, suggesting FGFR4 as a potential driver of endocrine resistance. In this study, we investigated the role of FGFR4 in mediating endocrine resistance and explored the therapeutic potential of targeting FGFR4 in advanced breast cancer. Methods: A gene expression signature of FGFR4 activity was examined in ER+ breast cancer pre- and post-neoadjuvant endocrine therapy and the association between FGFR4 expression and patient survival was examined. A correlation analysis was used to uncover potential regulators of FGFR4 overexpression. To investigate if FGFR4 is necessary to drive endocrine resistance, we tested response to FGFR4 inhibition in long term estrogen deprived (LTED) cells and their paired parental cells. Doxycycline inducible FGFR4 overexpression and knockdown cell models were generated to examine if FGFR4 was sufficient to confer endocrine resistance. Finally, we examined response to FGFR4 monotherapy or combination therapy with fulvestrant in breast cancer cell lines to explore the potential of FGFR4 targeted therapy for advanced breast cancer and assessed the importance of PAM50 subtype in response to FGFR4 inhibition. Results: A FGFR4 activity gene signature was significantly upregulated post neoadjuvant aromatase inhibitor treatment, and high FGFR4 expression predicted poorer survival in patients with ER+ breast cancer. Gene expression association analysis using TCGA, METABRIC and SCAN-B datasets uncovered ER as the most significant gene negatively correlated with FGFR4 expression. ER negatively regulates FGFR4 expression at both the mRNA and protein level across multiple ER+ breast cancer cell lines. Despite robust overexpression of FGFR4, LTED cells did not show enhanced responses to FGFR4 inhibition compared to parental cells. Similarly, FGFR4 overexpression, knockdown or hotspot mutations did not significantly alter response to endocrine treatment in ER+ cell lines, nor did FGFR4 and fulvestrant combination treatment show synergistic effects. The HER2-like subtype of breast cancer showed elevated expression of FGFR4 and an increased response to FGFR4 inhibition relative to other breast cancer subtypes. Conclusions: Despite ER-mediated upregulation of FGFR4 post endocrine therapy, our study does not support a general role of FGFR4 in mediating endocrine resistance in ER+ breast cancer. Our data suggests that specific genomic backgrounds such as HER2 expression may be required for FGFR4 function in breast cancer and should be further explored.

The EstroGene Database Reveals Diverse Temporal, Context-Dependent, and Bidirectional Estrogen Receptor Regulomes in Breast Cancer.

As one of the most successful cancer therapeutic targets, estrogen receptor-α (ER/ESR1) has been extensively studied over the past few decades. Sequencing technological advances have enabled genome-wide analysis of ER action. However, comparison of individual studies is limited by different experimental designs, and few meta-analyses are available. Here, we established the EstroGene database through unified processing of data from 246 experiments including 136 transcriptomic, cistromic, and epigenetic datasets focusing on estradiol (E2)-triggered ER activation across 19 breast cancer cell lines. A user-friendly browser (https://estrogene.org/) was generated for multiomic data visualization involving gene inquiry under user-defined experimental conditions and statistical thresholds. Notably, annotation of metadata associated with public datasets revealed a considerable lack of experimental details. Comparison of independent RNA-seq or ER ChIP-seq data with the same design showed large variability and only strong effects could be consistently detected. Temporal estrogen response metasignatures were defined, and the association of E2 response rate with temporal transcriptional factors, chromatin accessibility, and heterogeneity of ER expression was evaluated. Unexpectedly, harmonizing 146 E2-induced transcriptomic datasets uncovered a subset of genes harboring bidirectional E2 regulation, which was linked to unique transcriptional factors and highly associated with immune surveillance in the clinical setting. Furthermore, the context dependent E2 response programs were characterized in MCF7 and T47D cell lines, the two most frequently used models in the EstroGene database. Collectively, the EstroGene database provides an informative and practical resource to the cancer research community to uniformly evaluate key reproducible features of ER regulomes and unravels modes of ER signaling. SIGNIFICANCE: A resource database integrating 246 publicly available ER profiling datasets facilitates meta-analyses and identifies estrogen response temporal signatures, a bidirectional program, and model-specific biases.

EstroGene database reveals diverse temporal, context-dependent and directional estrogen receptor regulomes in breast cancer.

As one of the most successful cancer therapeutic targets, estrogen receptor-α (ER/ESR1) has been extensively studied in decade-long. Sequencing technological advances have enabled genome-wide analysis of ER action. However, reproducibility is limited by different experimental design. Here, we established the EstroGene database through centralizing 246 experiments from 136 transcriptomic, cistromic and epigenetic datasets focusing on estradiol-treated ER activation across 19 breast cancer cell lines. We generated a user-friendly browser ( https://estrogene.org/ ) for data visualization and gene inquiry under user-defined experimental conditions and statistical thresholds. Notably, documentation-based meta-analysis revealed a considerable lack of experimental details. Comparison of independent RNA-seq or ER ChIP-seq data with the same design showed large variability and only strong effects could be consistently detected. We defined temporal estrogen response metasignatures and showed the association with specific transcriptional factors, chromatin accessibility and ER heterogeneity. Unexpectedly, harmonizing 146 transcriptomic analyses uncovered a subset of E2-bidirectionally regulated genes, which linked to immune surveillance in the clinical setting. Furthermore, we defined context dependent E2 response programs in MCF7 and T47D cell lines, the two most frequently used models in the field. Collectively, the EstroGene database provides an informative resource to the cancer research community and reveals a diverse mode of ER signaling.

Transcriptional landscapes of de novo root regeneration from detached Arabidopsis leaves revealed by time-lapse and single-cell RNA sequencing analyses.

Detached Arabidopsis thaliana leaves can regenerate adventitious roots, providing a platform for studying de novo root regeneration (DNRR). However, the comprehensive transcriptional framework of DNRR remains elusive. Here, we provide a high-resolution landscape of transcriptome reprogramming from wound response to root organogenesis in DNRR and show key factors involved in DNRR. Time-lapse RNA sequencing (RNA-seq) of the entire leaf within 12 h of leaf detachment revealed rapid activation of jasmonate, ethylene, and reactive oxygen species (ROS) pathways in response to wounding. Genetic analyses confirmed that ethylene and ROS may serve as wound signals to promote DNRR. Next, time-lapse RNA-seq within 5 d of leaf detachment revealed the activation of genes involved in organogenesis, wound-induced regeneration, and resource allocation in the wounded region of detached leaves during adventitious rooting. Genetic studies showed that BLADE-ON-PETIOLE1/2, which control aboveground organs, PLETHORA3/5/7, which control root organogenesis, and ETHYLENE RESPONSE FACTOR115, which controls wound-induced regeneration, are involved in DNRR. Furthermore, single-cell RNA-seq data revealed gene expression patterns in the wounded region of detached leaves during adventitious rooting. Overall, our study not only provides transcriptome tools but also reveals key factors involved in DNRR from detached Arabidopsis leaves.

FGFR4: A promising therapeutic target for breast cancer and other solid tumors.

The fibroblast growth factor receptor (FGFR) signaling pathway has long been known to cancer researchers because of its role in cell survival, proliferation, migration, and angiogenesis. Dysregulation of FGFR signaling is frequently reported in cancer studies, but most of these studies focus on FGFR1-3. However, there is growing evidence implicating an important and unique role of FGFR4 in oncogenesis, tumor progression, and resistance to anti-tumor therapy in multiple types of cancer. Importantly, there are several novel FGFR4-specific inhibitors in clinical trials, making FGFR4 an attractive target for further research. In this review, we focus on assessing the role of FGFR4 in cancer, with an emphasis on breast cancer. First, the structure, physiological functions and downstream signaling pathways of FGFR4 are introduced. Next, different mechanisms reported to cause aberrant FGFR4 activation and their functions in cancer are discussed, including FGFR4 overexpression, FGF ligand overexpression, FGFR4 somatic hotspot mutations, and the FGFR4 G388R single nucleotide polymorphism. Finally, ongoing and recently completed clinical trials targeting FGFRs in cancer are reviewed, highlighting the therapeutic potential of FGFR4 inhibition for the treatment of breast cancer.

Single Cell Analysis of Endothelial Cells Identified Organ-Specific Molecular Signatures and Heart-Specific Cell Populations and Molecular Features.

Endothelial cells line the inner surface of vasculature and play an important role in normal physiology and disease progression. Although most tissue is known to have a heterogeneous population of endothelial cells, transcriptional differences in organ specific endothelial cells have not been systematically analyzed at the single cell level. The Tabula Muris project profiled mouse single cells from 20 organs. We found 10 of the organs profiled by this Consortium have endothelial cells. Unsupervised analysis of these endothelial cells revealed that they were mainly grouped by organs, and organ-specific cells were further partially correlated by germ layers. Unexpectedly, we found all lymphatic endothelial cells grouped together regardless of their resident organs. To further understand the cellular heterogeneity in organ-specific endothelial cells, we used the heart as an example. As a pump of the circulation system, the heart has multiple types of endothelial cells. Detailed analysis of these cells identified an endocardial endothelial cell population, a coronary vascular endothelial cell population, and an aorta-specific cell population. Through integrated analysis of the single cell data from another two studies analyzing the aorta, we identified conserved cell populations and molecular markers across the datasets. In summary, by reanalyzing the existing endothelial cell single-cell data, we identified organ-specific molecular signatures and heart-specific subpopulations and molecular markers. We expect these findings will pave the way for a deeper understanding of vascular biology and endothelial cell-related diseases.

Control of de novo root regeneration efficiency by developmental status of Arabidopsis leaf explants.

De novo root regeneration (DNRR) has wide applications in agriculture such as those related to cutting technology. Detached Arabidopsis thaliana leaf explants can regenerate adventitious roots without added hormones. The regenerative ability is highly dependent on the developmental status of the leaf. An immature leaf has a higher regenerative ability, while a mature leaf is difficult to regenerate. Using RNA-Seq analysis, we showed that the expression levels of many genes, including those in the auxin network, changed during leaf maturation. Particularly, the expression levels of many YUCCA (YUC) genes in the auxin biosynthesis pathway are responsive to leaf maturation. Overexpression of YUC1 in the yuc-1D dominant mutant rescued the rooting defects caused by leaf maturation. In addition, YUC4 expression levels were also affected by circadian rhythms. The regenerative ability was reduced in both immature and mature mutant leaf explants from the new wuschel-related homeobox 11-3 (wox11-3) and wox12-3 mutant alleles created by the CRISPR/Cas9 method. Overall, the transcriptome and genetic data, together with the auxin concentration analysis, indicate that the ability to upregulate auxin levels upon detachment may be reduced during leaf maturation. Thus, multiple developmental and environmental signals may converge to control auxin accumulation, which affects the efficiency of the WOX11/12-mediated DNRR from leaf explants.

Jasmonate-mediated wound signalling promotes plant regeneration.

Wounding is the first event triggering regeneration1-4. However, the molecular basis of wound signalling pathways in plant regeneration is largely unclear. We previously established a method to study de novo root regeneration (DNRR) in Arabidopsis thaliana5,6, which provides a platform for analysing wounding. During DNRR, auxin is biosynthesized after leaf detachment and promotes cell fate transition to form the root primordium5-7. Here, we show that jasmonates (JAs) serve as a wound signal during DNRR. Within 2 h of leaf detachment, JA is produced in leaf explants and activates ETHYLENE RESPONSE FACTOR109 (ERF109). ERF109 upregulates ANTHRANILATE SYNTHASE α1 (ASA1)-a tryptophan biosynthesis gene in the auxin production pathway8-10-dependent on the pre-deposition of SET DOMAIN GROUP8 (SDG8)-mediated histone H3 lysine 36 trimethylation (H3K36me3)11 on the ASA1 locus. After 2 h, ERF109 activity is inhibited by direct interaction with JASMONATE-ZIM-DOMAIN (JAZ) proteins to prevent hypersensitivity to wounding. Our results suggest that a dynamic JA wave cooperates with histone methylation to upregulate a pulse of auxin production and promote DNRR in response to wounding.

Wound signaling: The missing link in plant regeneration.

Wounding is the first event that occurs in plant regeneration. However, wound signaling in plant regeneration is barely understood. Using a simple system of de novo root organogenesis from Arabidopsis thaliana leaf explants, we analyzed the genes downstream of wound signaling. Leaf explants may produce at least two kinds of wound signals to trigger short-term and long-term wound signaling. Short-term wound signaling is primarily involved in controlling auxin behavior and the fate transition of regeneration-competent cells, while long-term wound signaling mainly modulates the cellular environment at the wound site and maintains the auxin level in regeneration-competent cells. YUCCA (YUC) genes, which are involved in auxin biogenesis, are targets of short-term wound signaling in mesophyll cells and of long-term wound signaling in regeneration-competent cells. The expression patterns of YUCs provide important information about the molecular basis of wound signaling in plant regeneration.

YUCCA-mediated auxin biogenesis is required for cell fate transition occurring during de novo root organogenesis in Arabidopsis.

Many plant organs have the ability to regenerate a new plant after detachment or wounding via de novo organogenesis. During de novo root organogenesis from Arabidopsis thaliana leaf explants, endogenic auxin is essential for the fate transition of regeneration-competent cells to become root founder cells via activation of WUSCHEL-RELATED HOMEOBOX 11 (WOX11). However, the molecular events from leaf explant detachment to auxin-mediated cell fate transition are poorly understood. In this study, we used an assay to determine the concentration of indole-3-acetic acid (IAA) to provide direct evidence that auxin is produced after leaf explant detachment, a process that involves YUCCA (YUC)-mediated auxin biogenesis. Inhibition of YUC prevents expression of WOX11 and fate transition of competent cells, resulting in the blocking of rooting. Further analysis showed that YUC1 and YUC4 act quickly (within 4 hours) in response to wounding after detachment in both light and dark conditions and promote auxin biogenesis in both mesophyll and competent cells, whereas YUC5, YUC8, and YUC9 primarily respond in dark conditions. In addition, YUC2 and YUC6 contribute to rooting by providing a basal auxin level in the leaf. Overall, our study indicates that YUC genes exhibit a division of labour during de novo root organogenesis from leaf explants in response to multiple signals.

Auxin-Independent NAC Pathway Acts in Response to Explant-Specific Wounding and Promotes Root Tip Emergence during de Novo Root Organogenesis in Arabidopsis.

Plants have powerful regenerative abilities that allow them to recover from damage and survive in nature. De novo organogenesis is one type of plant regeneration in which adventitious roots and shoots are produced from wounded and detached organs. By studying de novo root organogenesis using leaf explants of Arabidopsis (Arabidopsis thaliana), we previously suggested that wounding is the first event that provides signals to trigger the whole regenerative process. However, our knowledge of the role of wounding in regeneration remains limited. In this study, we show that wounding not only triggers the auxin-mediated fate transition of regeneration-competent cells, but also induces the NAC pathway for root tip emergence. The NAC1 transcription factor gene was specifically expressed in response to wounding in the leaf explant, but not in the wounded leaf residue of the source plant. Inhibition of the NAC1 pathway severely affected the emergence of adventitious root tips. However, the NAC1 pathway functioned independently of auxin-mediated cell fate transition and regulates expression of CEP genes, which encode proteins that might have a role in degradation of extensin proteins in the cell wall. Overall, our results suggest that wounding has multiple roles in de novo root organogenesis and that NAC1 acts as one downstream branch in regulating the cellular environment for organ emergence.