Contribution of vasculature to stem integrity in Arabidopsis thaliana.

In plants, coordinated growth is important for organ mechanical integrity because cells remain contiguous through their walls. So far, defects in inflorescence stem integrity in Arabidopsis thaliana have mainly been related to epidermal defects. Although these observations suggest a growth-limiting function at the stem cortex, deeper layers of the stem could also contribute to stem integrity. The nac secondary cell wall thickening promoting factor1 (nst1) nst3 double-mutant background is characterized by weaker vascular bundles without cracks. By screening for the cracking phenotype in this background, we identified a regulator of stem cracking, the transcription factor INDETERMINATE DOMAIN9 (IDD9). Stem cracking was not caused by vascular bundle breakage in plants that expressed a dominant repressor version of IDD9. Instead, cracking emerged from increased cell expansion in non-lignified interfascicular fiber cells that stretched the epidermis. This phenotype could be enhanced through CLAVATA3-dependent cell proliferation. Collectively, our results demonstrate that stem integrity relies on three additive mechanical components: the epidermis, which resists inner cell growth; cell proliferation in inner tissues; and growth heterogeneity associated with vascular bundle distribution in deep tissues.

Stem integrity in Arabidopsis thaliana requires a load-bearing epidermis.

Because plant cells are glued to each other via their cell walls, failure to coordinate growth among adjacent cells can create cracks in tissues. Here, we find that the unbalanced growth of inner and outer tissues in the clavata3 de-etiolated3 (clv3 det3) mutant of Arabidopsis thaliana stretched epidermal cells, ultimately generating cracks in stems. Stem growth slowed before cracks appeared along clv3 det3 stems, whereas inner pith cells became drastically distorted and accelerated their growth, yielding to stress, after the appearance of cracks. This is consistent with a key role of the epidermis in restricting growth. Mechanical property measurements recorded using an atomic force microscope revealed that epidermal cell wall stiffness decreased in det3 and clv3 det3 epidermises. Thus, we hypothesized that stem integrity depends on the epidermal resistance to mechanical stress. To formally test this hypothesis, we used the DET3 gene as part of a tissue-specific strategy to complement cell expansion defects. Epidermis-driven DET3 expression restored growth and restored the frequency of stem cracking to 20% of the clv3 det3 mutant, demonstrating the DET3-dependent load-bearing role of the epidermis.

Contributions of lignification, tissue arrangement patterns, and cross-sectional area to whole-stem mechanical properties in Arabidopsis thaliana.

Plant cells withstand mechanical stress originating from turgor pressure by robustly maintaining the mechanical properties of the cell wall. This applies at the organ scale as well; many plant stems act as pressurized cylinders, where the epidermis is under tension and inner tissues are under compression. The clavata3 de-etiolated3 (clv3-8 det3-1) double mutant of Arabidopsis thaliana displays cracks in its stems because of a conflict between the mechanical properties of the weak epidermis and over-proliferation of inner stem tissues. In this work, we conducted three-point bending tests on various Arabidopsis thaliana mutants, including those displaying the stem cracking phenotype, to examine the differences in their mechanical properties. The clv3-8 det3-1 double mutant exhibited reduced stem stiffness, consistent with reduced differentiation due to the clv3-8 mutation. Yet, in clv3-8, stem cross-sectional area was increased associating with the increase in vascular bundle number, and stem cross-sections displayed various shapes. To uncouple the contribution of geometry and cell-wall differentiation to the mechanical properties of the whole stems, we tested the contribution of lignified fibers to stem stiffness. In order to suppress lignin deposition in stems genetically, we generated multiple higher-order mutants by crossing clv3-8 and/or det3-1 with nst1-1 nst3-1, in which lignin deposition is suppressed. Stem stiffness was reduced markedly in all nst1-1 nst3-1 mutant backgrounds. Overall, our results suggest that stem stiffness relies on the presence of differentiated, lignified, fiber tissue as well as on the alignment or spatial distribution of vascular bundles within the stem organ.

An auxin signaling network translates low-sugar-state input into compensated cell enlargement in the fugu5 cotyledon.

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

Intratumoral Tumor Infiltrating Lymphocytes (TILs) are Associated With Cell Proliferation and Better Survival But Not Always With Chemotherapy Response in Breast Cancer.

OBJECTIVE: To investigate the clinical relevance of intratumoral tumor infiltrating lymphocytes (TILs) in breast cancer as measured by computational deconvolution of bulk tumor transcriptomes. SUMMARY BACKGROUND DATA: Commonly assessed TILs, located in tumor stroma without direct contact with cancer cells (stromal TILs), correlate with breast cancer treatment response and survival. The clinical relevance of intratumoral TILs has been less studied partly due to their rarity; however, they may have nonnegligible effects given their direct contact with cancer cells. METHODS: In all, 5870 breast cancer patients from TCGA, METABRIC, GSE96058, GSE25066, GSE163882, GSE123845, and GSE20271 cohorts were analyzed and validated. RESULTS: The intratumoral TIL score was established by the sum of all types of lymphocytes using the xCell algorithm. This score was the highest in triple-negative breast cancer (TNBC) and the lowest in the ER-positive/HER2-negative subtype. It correlated with cytolytic activity and infiltrations of dendritic cells, macrophages, and monocytes, and uniformly enriched immune-related gene sets regardless of subtype. Intratumoral TIL-high tumors correlated with higher mutation rates and significant cell proliferation on biological, pathological, and molecular analyses only in the ER-positive/HER2-negative subtype. It was significantly associated with pathological complete response after anthracycline- and taxane-based neoadjuvant chemotherapy in about half of the cohorts, regardless of the subtype. Intratumoral TIL-high tumors correlated with better overall survival in HER2-positive and TNBC subtypes consistently in 3 cohorts. CONCLUSIONS: Intratumoral TILs estimated by transcriptome computation were associated with increased immune response and cell proliferation in ER-positive/HER2-negative and better survival in HER2-positive and TNBC subtypes, but not always with pathological complete response after neoadjuvant chemotherapy.

Germinating seedlings and mature shoots of Cuscuta campestris respond differently to light stimuli during parasitism but not during circumnutation.

Seedlings of the parasitic plant genus Cuscuta (dodder) locate hosts by circumnutation, coil around the host near soil level and form a haustorium, establishing a primary parasitism beneath the canopy. Mature shoots elongating from the parasitic region parasitize other hosts on the upper surfaces of their canopy. Although parasitism by dodder is stimulated by blue and far-red light, and inhibited by red light, the responses to light signals during the developmental stages are not comprehensively understood. Therefore, we compared the effects of different types of light on both circumnutation and parasitism by germinating seedlings and mature shoots of Cuscuta campestris. Seedlings established parasitism under blue and far-red light, but not under red light, as has been reported repeatedly. By contrast, mature shoots exhibited coiling around the host and haustoria formation even under a red light as well as under blue and far-red light. These findings indicate that C. campestris modified its response to red light during the transition from young seedlings to mature shoots, facilitating parasitism. Light quality did not affect the circumnutation of either seedlings or mature shoots, indicating that circumnutation and the coiling movement that leads to parasitism were regulated by different environmental signals.

Intratumoral PDGFB gene predominantly expressed in endothelial cells is associated with angiogenesis and lymphangiogenesis, but not with metastasis in breast cancer.

PURPOSE: Platelet-derived growth factor B (PDGFB) is known to play essential roles in angiogenesis and lymphangiogenesis during development, and tumor growth and vessel stabilization in experimental models. However, whether these findings could be translated to breast cancer patients remains unclear. We hypothesized that PDGFB gene expression is associated with angiogenesis, cell proliferation, and clinical outcomes in breast cancer patients. METHODS: A total of 7635 primary breast cancer patients with full transcriptome and clinical data available from 13 independent cohorts were analyzed using in silico approach. The median value was used to divide each cohort into high and low PDGFB expression groups. RESULTS: High PDGFB gene expression was associated with increased expression of angiogenesis-related genes, higher amount of vascular cell infiltrations, and with enrichment of angiogenesis gene set, lymphangiogenesis-related gene expressions, lymphangiogenesis-related cell infiltrations, and enrichmentof lymphangiogenesis gene set in GSE96058 and validated by TCGA cohorts; however, not with lymphatic metastasis. PDGFB expression was neither associated with cell proliferation as assessed by Ki67 expression nor with Nottingham histological grade, or response to neoadjuvant chemotherapy. We found that PDGFB was most extensively expressed by endothelial and perivascular-like cells in the tumor microenvironment, and minimally by cancer cells consistently in two single-cell sequence cohorts. High PDGFB expression enriched TGFß, epithelial-mesenchymal transition, hypoxia, and cancer stem cell-associated pathways. However, no association with distant metastasis was observed. Disease-specific and disease-free survival were worse in the high PDGFB expression group consistently in TCGA and METABRIC cohorts. CONCLUSION: PDGFB is predominantly expressed in endothelial cells and is associated with angiogenesis and lymphangiogenesis, but not with cellular proliferation or metastasis in breast cancer.

High RAD51 gene expression is associated with aggressive biology and with poor survival in breast cancer.

PURPOSE: Although the DNA repair mechanism is important in preventing carcinogenesis, its activation in established cancer cells may support their proliferation and aggravate cancer progression. RAD51 cooperates with BRCA2 and is essential in the homologous recombination of DNA repair. To this end, we hypothesized that RAD51 gene expression is associated with cancer cell proliferation and poor prognosis of breast cancer (BC) patients. METHODS: A total of 8515 primary BC patients with transcriptome and clinical data from 17 independent cohorts were analyzed. The median value was used to divide each cohort into high and low RAD51 expression groups. RESULTS: High RAD51 expression enriched the DNA repair gene set and was correlated with DNA repair-related genes. Nottingham histological grade, Ki67 expression and cell proliferation-related gene sets (E2F Targets, G2M Checkpoint and Myc Targets) were all significantly associated with the high RAD51 BC group. RAD51 expression was positively correlated with Homologous Recombination Deficiency, as well as both mutational burden and neoantigens that accompanied a higher infiltration of immune cells. Primary BC with lymph node metastases was associated with high expression of RAD51 in two cohorts. There was no strong correlation between RAD51 expression and drug sensitivity in cell lines, and RAD51 expression was lower after the neoadjuvant chemotherapy compared to before the treatment. High RAD51 BC was associated with poor prognosis consistently in three independent cohorts. CONCLUSION: RAD51 gene expression is associated with aggressive cancer biology, cancer cell proliferation, and poor survival in breast cancer.

Differences in drug sensitivity between two-dimensional and three-dimensional culture systems in triple-negative breast cancer cell lines.

Three-dimensional (3D) culture reflects tumor biology complexities compared with two-dimensional (2D) culture. Thus, 3D culture has attracted attention in cell biology studies including drug sensitivity tests. Herein, we investigated differences in anticancer drug sensitivities between 2D and 3D culture systems in triple-negative breast cancer (TNBC) cell lines. Thirteen TNBC cell lines were maintained in 2D and 3D cultures for 3 days before drug exposure. Cell morphology in the 3D culture was examined by phase-contrast microscopy. Sensitivities to epirubicin (EPI), cisplatin (CDDP), and docetaxel (DTX) were investigated by cell viability assay in both cultures and compared. The IC50s of all 3 drugs were significantly higher in the 3D culture than in the 2D culture in most cell lines. Those were correlated between the 2D and 3D cultures in EPI (R = 0.555) and CDDP (R = 0.955), but not in DTX (R = 0.221). Round spheroid-forming cells were more resistant to agents than grape-like types. In conclusion, 3D culture was more resistant to all 3 drugs than 2D culture in most TNBC cell lines. Sensitivity to CDDP was highly correlated between the 2D and 3D cultures, but not to DTX. 2D culture may be acceptable for sensitivity test for DNA-damaging agents.

Lymphovascular invasion in breast cancer is associated with gene expression signatures of cell proliferation but not lymphangiogenesis or immune response.

BACKGROUND: While the prognostic relevance of lymphovascular invasion (LVI) in breast cancer is well known, its molecular biology is poorly understood. We hypothesized that pathologically determined LVI reflects molecular features of tumors and can be discerned from their genomic and transcriptomic profiles. METHODS: LVI status and Nottingham histological scores of primary breast tumors of The Cancer Genome Atlas (TCGA) project were assessed from pathology reports; other clinical and molecular data were obtained from TCGA data portals and publications. Two independent datasets (GSE5460 and GSE7849) were combined and used for validation. RESULTS: LVI status was determinable for 639 and 196 cases of the TCGA and validation cohorts, among whom LVI incidence was 37.8% and 37.2%, respectively. LVI was associated with high tumor Ki67 expression, advanced pathologic stage, and high Nottingham scores. LVI-positive cases had worse overall and progression-free survival regardless of cancer subtype. Surprisingly, in both cohorts, LVI was not associated with lymphangiogenesis or lymphatic vessel density as estimated from tumor expression of lymphatic endothelium-associated genes. LVI-positive tumors had higher genome copy number aberrations, aneuploidy, and homologous recombination defects, but not single-nucleotide variations or intra-tumor genome heterogeneity. Tumor immune cell composition and cytolytic activity was not associated with LVI status. On the other hand, expression of cell proliferation-related genes was significantly increased in LVI-positive tumors. CONCLUSION: Our study suggests that breast cancer with LVI is a highly proliferative cancer, and it does not correlate with gene expression markers for lymphangiogenesis or immune response.

Molecular Biological Features of Nottingham Histological Grade 3 Breast Cancers.

INTRODUCTION: Cancer biology dominates the behavior and prognosis of a tumor. Although Nottingham histological grade is a subjective pathological determination, it has been accepted as a surrogate model for cancer biology. As such, histologic grade was incorporated into the latest 8th edition of the American Joint Committee on Cancer breast cancer staging system. In this study, we hypothesized that grade 3 breast cancers demonstrate aggressive molecular biological profiles, reflecting worse biology and possible underlying immunogenicity. METHODS: Transcriptomic and clinical data were obtained from the Molecular Taxonomy of Breast Cancer International Consortium, and the findings were validated by The Cancer Genome Atlas breast cancer cohort and GSE25066. RESULTS: Overall, 2876 patients were analyzed in this study. Grade 3 tumors were more common in estrogen receptor (ER)-negative, advanced-stage patients, and were associated with human epidermal growth factor receptor 2 and basal subtypes by the PAM50 classifier, as well as with increased MKI67 expression (all p <0.001). Disease-free survival was significantly worse in grade 3 tumors (all cohorts). Gene set enrichment analysis demonstrated that grade 3 tumors were significantly enriched with not only cell proliferation and cell cycle-related gene sets but also immune activity-related gene sets. CIBERSORT confirmed that grade 3 tumors were infiltrated with macrophage M1, follicular helper T cells, and activated natural killer cells (all p <0.001). Furthermore, grade 3 tumors were associated with more diverse T cell receptors (p =0.001) and increased cytolytic activity (p <0.001). Lastly, major T-cell exhaustion markers were significantly elevated in grade 3 breast cancers (p <0.001). CONCLUSION: Grade 3 breast cancers demonstrated aggressive transcriptomic features with enhanced immunogenicity and elevated T-cell exhaustion markers.

CD8 T Cell Score as a Prognostic Biomarker for Triple Negative Breast Cancer.

CD8 T cell is an essential component of tumor-infiltrating lymphocytes (TIL) and tumor immune microenvironment (TIME). Using the xCell CD8 T cell score of whole tumor gene expression data, we estimated these cells in total of 3837 breast cancer patients from TCGA, METABRIC and various GEO cohorts. The CD8 score correlated strongly with expression of CD8 genes. The score was highest for triple-negative breast cancer (TNBC), and a high score was associated with high tumor immune cytolytic activity and better survival in TNBC but not other breast cancer subtypes. In TNBC, tumors with a high CD8 score had enriched expression of interferon (IFN)-α and IFN-γ response and allograft rejection gene sets, and greater infiltration of anti-cancerous immune cells. The score strongly correlated with CD4 memory T cells in TNBC, and tumors with both a high CD8 score and high CD4 memory T cell abundance had significantly better survival. Finally, a high CD8 score was significantly associated with high expression of multiple immune checkpoint molecules. In conclusion, a high CD8 T cell score is associated with better survival in TNBC, particularly when tumor CD4 memory T cells were elevated. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy.

High Expression of microRNA-143 is Associated with Favorable Tumor Immune Microenvironment and Better Survival in Estrogen Receptor Positive Breast Cancer.

microRNA-143(miR-143) is a well-known tumor suppressive microRNA that exhibits anti-tumoral function by targeting KRAS signaling pathways in various malignancies. We hypothesized that miR-143 suppresses breast cancer progression by targeting KRAS and its effector molecules. We further hypothesized that high expression of miR-143 is associated with a favorable tumor immune microenvironment of estrogen receptor (ER)-positive breast cancer patients which result in improved survival. Two major publicly available breast cancer cohorts; The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) were used. The miR-143 high expression group was associated with increased infiltration of anti-cancer immune cells and decreased pro-cancer immune cells, as well as enrichment of the genes relating to T helper (Th1) cells resulting in improved overall survival (OS) in ER-positive breast cancer patients. To the best of our knowledge, this is the first study to demonstrate that high expression of miR-143 in cancer cells associates with a favorable tumor immune microenvironment, upregulation of anti-cancer immune cells, and suppression of the pro-cancer immune cells, associating with better survival of the breast cancer patients.

Excess Pyrophosphate within Guard Cells Delays Stomatal Closure.

A variety of cellular metabolic reactions generate inorganic pyrophosphate (PPi) as an ATP hydrolysis byproduct. The vacuolar H+-translocating pyrophosphatase (H+-PPase) loss-of-function fugu5 mutant is susceptible to drought and displays pleotropic postgerminative growth defects due to excess PPi. It was recently reported that stomatal closure after abscisic acid (ABA) treatment is delayed in vhp1-1, a fugu5 allele. In contrast, we found that specific removal of PPi rescued all of the above fugu5 developmental and growth defects. Hence, we speculated that excess PPi itself, rather than vacuolar acidification, might delay stomatal closure. To test this hypothesis, we constructed transgenic plants expressing the yeast IPP1 gene (encoding a cytosolic pyrophosphatase) driven by a guard cell-specific promoter (pGC1::IPP1) in the fugu5 background. Our measurements confirmed stomatal closure defects in fugu5, further supporting a role for H+-PPase in stomatal functioning. Importantly, while pGC1::IPP1 transgenics morphologically mimicked fugu5, stomatal closure was restored in response to ABA and darkness. Quantification of water loss revealed that fugu5 stomata were almost completely insensitive to ABA. In addition, growth of pGC1::IPP1 plants was promoted compared to fugu5 throughout their life; however, it did not reach the wild type level. fugu5 also displayed an increased stomatal index, in violation of the one-cell-spacing rule, and phenotypes recovered upon removal of PPi by pAVP1::IPP1 (FUGU5, VHP1 and AVP1 are the same gene encoding H+-PPase), but not in the pGC1::IPP1 line. Taken together, these results clearly support our hypothesis that dysfunction in stomata is triggered by excess PPi within guard cells, probably via perturbed guard cell metabolism.

Tumor Heterogeneity Correlates with Less Immune Response and Worse Survival in Breast Cancer Patients.

BACKGROUND: Intratumor heterogeneity implies that subpopulations of cancer cells that differ in genetic, phenotypic, or behavioral characteristics coexist in a single tumor (Ma in Breast Cancer Res Treat 162(1):39-48, 2017; Martelotto in Breast Cancer Res 16(3):210, 2014). Tumor heterogeneity drives progression, metastasis and treatment resistance, but its relationship with tumor infiltrating immune cells is a matter of debate, where some argue that tumors with high heterogeneity may generate neoantigens that attract immune cells, and others claim that immune cells provide selection pressure that shapes tumor heterogeneity (McGranahan et al. in Science 351(6280):1463-1469, 2016; McGranahan and Swanton in Cell 168(4):613-628, 2017). We sought to study the association between tumor heterogeneity and immune cells in a real-world cohort utilizing The Cancer Genome Atlas. METHODS: Mutant allele tumor heterogeneity (MATH) was calculated to estimate intratumoral heterogeneity, and immune cell compositions were estimated using CIBERSORT. Survival analyses were demonstrated using Kaplan-Meir curves. RESULTS: Tumors with high heterogeneity (high MATH) were associated with worse overall survival (p = 0.049), as well as estrogen receptor-positive (p = 0.011) and non-triple-negative tumors (p = 0.01). High MATH tumors were also associated with less infiltration of anti-tumor CD8 (p < 0.013) and CD4 T cells (p < 0.00024), more tumor-promoting regulatory T cells (p < 4e-04), lower expression of T-cell exhaustion markers, specifically PDL-1 (p = 0.0031), IDO2 (p = 0.34), ADORA2A (p = 0.018), VISTA (p = 0.00013), and CCR4 (p < 0.00001), lower expression of cytolytic enzymes granzyme A (p = 0.0056) and perforin 1 (p = 0.053), and low cytolytic activity score (p = 0.0028). CONCLUSIONS: High heterogeneity tumors are associated with less immune cell infiltration, less activation of the immune response, and worse survival in breast cancer. Our results support the notion that tumor heterogeneity is shaped by selection pressure of tumor-infiltrating immune cells.

APOBEC3-Mediated RNA Editing in Breast Cancer is Associated with Heightened Immune Activity and Improved Survival.

APOBEC3 enzymes contribute significantly to DNA mutagenesis in cancer. These enzymes are also capable of converting C bases at specific positions of RNAs to U. However, the prevalence and significance of this C-to-U RNA editing in any cancer is currently unknown. We developed a bioinformatics workflow to determine RNA editing levels at known APOBEC3-mediated RNA editing sites using exome and mRNA sequencing data of 1040 breast cancer tumors. Although reliable editing determinations were limited due to sequencing depth, editing was observed in both tumor and adjacent normal tissues. For 440 sites (411 genes), editing was determinable for ≥5 tumors, with editing occurring in 0.6%-100% of tumors (mean 20%, SD 14%) at an average level of 0.6%-20% (mean 7%, SD 4%). Compared to tumors with low RNA editing, editing-high tumors had enriched expression of immune-related gene sets, and higher T cell and M1 macrophage infiltration, B and T cell receptor diversity, and immune cytolytic activity. Concordant with this, patients with increased RNA editing in tumors had better disease- and progression-free survivals (hazard ratio = 1.67-1.75, p < 0.05). Our study identifies that APOBEC3-mediated RNA editing occurs in breast cancer tumors and is positively associated with elevated immune activity and improved survival.

Estrogen Receptor Positive Breast Cancer with High Expression of Androgen Receptor has Less Cytolytic Activity and Worse Response to Neoadjuvant Chemotherapy but Better Survival.

Estrogen receptor (ER) positive breast cancer (BC), the most abundant BC subtype, is notorious for poor response to neoadjuvant chemotherapy (NAC). The androgen receptor (AR) was reported to support estradiol-mediated ER activity in an in vitro system. Recently, ER-positive BC with fewer tumor infiltrating lymphocytes (TILs) was shown to have a better prognosis, opposite to the trend seen with ER-negative BC. We hypothesized that ER-positive BC with high expression of AR will have fewer TILs and an inferior response to NAC, but with a better prognosis. In both TCGA and METABRIC cohorts, AR expression was significantly higher in ER-positive BCs compared to ER-negatives (p < 0.001, p < 0.001, respectively) and it correlated with ER expression (R = 0.630, R = 0.509, respectively). In ER-positive tumors, AR high tumors enriched UV response down (NES = 2.01, p < 0.001), and AR low tumors enriched DNA repair (NES = -2.02, p < 0.001). AR high tumors were significantly associated with procancer regulatory T-cells, and AR low tumors were associated with anticancer immune cells, such as CD4, CD8, and Gamma-Delta T-cells and memory B-cells in ER-positive BC (p < 0.01). Further, cytolytic activity was significantly lower in AR high BC in both cohorts. Finally, AR high tumors had a significantly lower rate of attaining pathological complete response to NAC (GSE22358), but better survival. In conclusion, our results demonstrated that high AR has fewer tumor infiltrating lymphocytes as well as cytolytic activity and an inferior response to NAC, but better survival in ER-positive BC.

Biochemical, Structural and Physiological Characteristics of Vacuolar H+-Pyrophosphatase.

Proton-translocating inorganic pyrophosphatase (H+-PPase) actively translocates protons across membranes coupled with the hydrolysis of inorganic pyrophosphate (PPi). H+-PPase, which is composed of a single protein and uses a simple compound as a substrate, has been recognized as a new type of ion pump in addition to the P-, F- and V-type ion-translocating ATPases. H+- and Na+-PPases are distributed in various organisms including plants, parasitic protozoa, Archaebacteria and bacteria, but are not present in animals or yeast. Vacuolar H+-PPase has dual functions in plant cells: hydrolysis of cytosolic PPi to maintain the levels of PPi, and translocation of protons into vacuoles to maintain the acidity of the vacuolar lumen. Acidification performed with the vacuolar-type H+-ATPase and H+-PPase is essential to maintain acidic conditions, which are necessary for vacuolar hydrolytic enzymes and for supplying energy to secondary active transporters. Recent studies using loss-of-function mutant lines of H+-PPase and complementation lines with soluble PPases have emphasized the physiological importance of the scavenging role of PPi. An overview of the main features of H+-PPases present in the vacuolar membrane is provided in terms of tissue distribution in plants, intracellular localization, structure-function relationship, biochemical potential as a proton pump and functional stability.

Compensated Cell Enlargement in fugu5 is Specifically Triggered by Lowered Sucrose Production from Seed Storage Lipids.

To reveal the logic of size regulation in multicellular organisms, we have used Arabidopsis thaliana as a model organism and its leaves as a model organ. We discovered the existence of a compensatory system, whereby a decrease in leaf cell number often triggers unusual cell enlargement. However, despite the large number of compensation-exhibiting mutants analyzed to date, we have only a limited understanding of the detailed molecular mechanisms triggering the decrease in cell number and subsequent compensated cell enlargement (CCE). CCE in fugu5, the vacuolar type H+-pyrophosphatase loss-of-function mutant, is specific to cotyledons and completely suppressed when sucrose (Suc) is supplied or cytosolic pyrophosphate (PPi) is specifically removed. In addition, several lines of evidence suggest that excess cytosolic PPi in fugu5 impairs gluconeogenesis from triacylglycerol (TAG) to Suc. Here, detailed cellular phenotyping revealed that the loss-of-function mutants icl-2, mls-2 and pck1-2 triggered CCE in cotyledons. All double mutant combinations between fugu5-1 and the above three mutants exhibited compensation, but did not display a further increase in cell size. Importantly, similar phenotypes were observed in icl-2 mls-2, icl-2 pck1-2 and mls-2 pck1-2. Quantification of TAG breakdown and Suc contents further supported our findings. Taken together, we demonstrate that de novo Suc synthesis from TAG is fundamentally important for proper resumption of post-germinative cotyledon development. Moreover, provided that icl-2, mls-2 and pck1-2 are only compromised in Suc biosynthesis de novo from TAG, our findings clearly indicate that lowered Suc production in fugu5, rather than excess cytosolic PPi, is the direct trigger of CCE.

Dynamics of vacuoles and H+-pyrophosphatase visualized by monomeric green fluorescent protein in Arabidopsis: artifactual bulbs and native intravacuolar spherical structures.

We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H(+)-pyrophosphatase (H(+)-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H(+)-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H(+)-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H(+)-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H(+)-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.