EGFR signaling controls directionality of epithelial multilayer formation upon loss of cell polarity.

Apical-basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, we found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc-finger transcription factor. Additionally, we identified Rho kinase as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation.

Cell polarity opposes Jak/STAT-mediated Escargot activation that drives intratumor heterogeneity in a Drosophila tumor model.

In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, we could detect and further validate the expression of Snail family transcription factor Escargot (Esg). We also ascertain a similar relationship between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, our results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity.

Single-cell transcriptomics identifies Keap1-Nrf2 regulated collective invasion in a Drosophila tumor model.

Apicobasal cell polarity loss is a founding event in epithelial-mesenchymal transition and epithelial tumorigenesis, yet how pathological polarity loss links to plasticity remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, we performed single-cell RNA sequencing of Drosophila ovaries, where inducing polarity-gene l(2)gl-knockdown (Lgl-KD) causes invasive multilayering of the follicular epithelia. Analyzing the integrated Lgl-KD and wildtype transcriptomes, we discovered the cells specific to the various discernible phenotypes and characterized the underlying gene expression. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Ectopic expression of Keap1 increased the volume of delaminated follicle cells that showed enhanced invasive behavior with significant changes to the cytoskeleton. Overall, our findings describe the comprehensive transcriptome of cells within the follicle cell tumor model at the single-cell resolution and identify a previously unappreciated link between Keap1-Nrf2 signaling and cell plasticity at early tumorigenesis.

In the body, most cells exhibit some form of spatial asymmetry: the compartments within the cell are not evenly distributed, thereby allowing the cells to know whether a surface is on the 'outside' or the 'inside' of a tissue or organ. In the cells of epithelial tissues, which line most of the cavities and the organs in the body, this asymmetry is known as apical-basal polarity. Maintaining apical-basal polarity in epithelial cells is one of the main barriers that stops cancer cells from invading other tissues, which is the first step of metastasis, the process through which cancer cells leave their tissue of our origin and spread to distant locations in the body. In the fruit fly Drosophila melanogaster, scientists have engineered cells in several tissues to stop producing the proteins that help establish apical-basal polarity, in an effort to study the earliest steps of tumor formation. Unfortunately, these experiments frequently lead to rampant metastasis, making it difficult to identify the earliest changes that make the tumor cells more likely to become invasive. Therefore, finding a tissue in which loss of apical-basal polarity does not cause aggressive cancer progression is necessary to address this gap in knowledge. The epithelial cell layer lining the ovaries of fruit flies may be such a tissue. When these cells lose their apical-basal polarity, rather than becoming metastatic and spreading to distant organs, they interleave with each other, forming a tumorous growth that only invades into the neighboring compartment. Chatterjee et al. used this system to study individual invasive cells. They wanted to know whether the genes that these cells switch on and off are known to be involved in human cancers, and if so, which of them control the invasive behavior of tumor cells. Chatterjee et al. determined that when cells in the fruit-fly ovary lost their polarity, they turned genes on and off in a pattern similar to that seen both in mammalian cancers and in tumors from other fly tissues. One of the notable changes they observed in the ovarian cells that lost apical-basal polarity was the activation of the Keap1/Nrf2 oxidative-stress signaling pathway, which normally protects cells from damage caused by excessive oxidation. In the ovarian cells, however, the activation of these genes also led to aggressive invasion of the collective tumor cells into the neighboring compartment. Interestingly, this increase in invasiveness was characterized by polarized changes within the cells, specifically in the scaffolding that allows cells to keep their shape and move: the edge of the cells leading the invasion had greater levels of a protein called actin, which enables the cells to protrude into the neighboring compartments. Chatterjee et al. have identified a new mechanism that impacts the migratory behavior of cells. Insights from their findings will pave the way for a better understanding of how and when this mechanism plays a role in metastasis.

Developmental regulation of epithelial cell cuboidal-to-squamous transition in Drosophila follicle cells.

Epithelial cells form continuous membranous structures for organ formation, and these cells are classified into three major morphological categories: cuboidal, columnar, and squamous. It is crucial that cells transition between these shapes during the morphogenetic events of organogenesis, yet this process remains poorly understood. All three epithelial cell shapes can be found in the follicular epithelium of Drosophila egg chamber during oogenesis. Squamous cells (SCs) are initially restricted to the anterior terminus in cuboidal shape. They then rapidly become flattened to assume squamous shape by stretching and expansion in 12 â€‹h during midoogenesis. Previously, we reported that Notch signaling activated a zinc-finger transcription factor Broad (Br) at the end of early oogenesis. Here we report that ecdysone and JAK/STAT pathways subsequently converge on Br to serve as an important spatiotemporal regulator of this dramatic morphological change of SCs. The early uniform pattern of Br in the follicular epithelium is directly established by Notch signaling at stage 5 of oogenesis. Later, ecdysone and JAK/STAT signaling activities synergize to suppress Br in SCs from stage 8 to 10a, contributing to proper SC squamous shape. During this process, ecdysone signaling is essential for SC stretching, while JAK/STAT regulates SC clustering and cell fate determination. This study reveals an inhibitory role of ecdysone signaling in suppressing Br in epithelial cell remodeling. In this study we also used single-cell RNA sequencing data to highlight the shift in gene expression which occurs as Br is suppressed and cells become flattened.

Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut.

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. Here, we show that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)-Ras signaling. We also show that ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, we find that the dividing enteroblasts do not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, our work uncovers a new role for EGFR-Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates.

Xenograft cancer vaccines prepared from immunodeficient mice increase tumor antigen diversity and host T cell efficiency against colorectal cancers.

Autologous cancer vaccines (ACVs) are a desirable approach for personalized medicine, but the efficiency of ACVs remains unsatisfactory due to their low immunogenicity. This study developed a platform that can enhance the immunogenicity of ACVs by transplanting the tumors into immunodeficient mice. The CT26 cell line was inoculated into severe combined immunodeficient mice (SCID) for vaccine preparation where escalates tumor development, subsequently diversifying the tumor antigenic topology. CT26/SCID cancer vaccines significantly inhibited tumor growth, increased the amount of tumor infiltrating lymphocytes, and triggered Th-1 predominant immune responses. Tumor antigenic profiles of CT26/SCID cells were further analyzed by liquid chromatography-tandem mass spectrometry. Compared to CT26 parental cells, a total of 428 differentially expressed proteins (DEPs) were detected. These DEPs revealed that CT26/SCID cells overexpressed several novel therapeutic targets, including KNG1, apoA-I and, ß2-GPI, which can trigger cytotoxic T cells towards Th-1 predominant immune responses and directly suppress proliferation in tumors. CT26/SCID cancer vaccines can be easily manufactured, while traits of triggering stronger antigen-specific Th-1 immune activity against tumors, are retained. Results of this study provide an effective proof-of-concept of an ACV for personalized cancer immunotherapy.

Modeling Notch-Induced Tumor Cell Survival in the Drosophila Ovary Identifies Cellular and Transcriptional Response to Nuclear NICD Accumulation.

Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact of NICD accumulation during tumorigenesis, we developed a tumor model using the Drosophila ovarian follicular epithelium. Using this system we demonstrated that NICD accumulation contributed to larger tumor growth, reduced apoptosis, increased nuclear size, and fewer incidents of DNA damage without altering ploidy. Using bulk RNA sequencing we identified key genes involved in both a pre- and post- tumor response to NICD accumulation. Among these are genes involved in regulating double-strand break repair, chromosome organization, metabolism, like raptor, which we experimentally validated contributes to early Notch-induced tumor growth. Finally, using single-cell RNA sequencing we identified follicle cell-specific targets in NICD-overexpressing cells which contribute to DNA repair and negative regulation of apoptosis. This valuable tumor model for nuclear NICD accumulation in adult Drosophila follicle cells has allowed us to better understand the specific contribution of nuclear NICD accumulation to cell survival in tumorigenesis and tumor progression.

Telomere Attrition and Clonal Hematopoiesis of Indeterminate Potential in Cardiovascular Disease.

Clinical evidence suggests that conventional cardiovascular disease (CVD) risk factors cannot explain all CVD incidences. Recent studies have shown that telomere attrition, clonal hematopoiesis of indeterminate potential (CHIP), and atherosclerosis (telomere-CHIP-atherosclerosis, TCA) evolve to play a crucial role in CVD. Telomere dynamics and telomerase have an important relationship with age-related CVD. Telomere attrition is associated with CHIP. CHIP is commonly observed in elderly patients. It is characterized by an increase in blood cell clones with somatic mutations, resulting in an increased risk of hematological cancer and atherosclerotic CVD. The most common gene mutations are DNA methyltransferase 3 alpha (DNMT3A), Tet methylcytosine dioxygenase 2 (TET2), and additional sex combs-like 1 (ASXL1). Telomeres, CHIP, and atherosclerosis increase chronic inflammation and proinflammatory cytokine expression. Currently, their epidemiology and detailed mechanisms related to the TCA axis remain incompletely understood. In this article, we reviewed recent research results regarding the development of telomeres and CHIP and their relationship with atherosclerotic CVD.

Polyploid mitosis and depolyploidization promote chromosomal instability and tumor progression in a Notch-induced tumor model.

Ploidy variation is a cancer hallmark and is frequently associated with poor prognosis in high-grade cancers. Using a Drosophila solid-tumor model where oncogenic Notch drives tumorigenesis in a transition-zone microenvironment in the salivary gland imaginal ring, we find that the tumor-initiating cells normally undergo endoreplication to become polyploid. Upregulation of Notch signaling, however, induces these polyploid transition-zone cells to re-enter mitosis and undergo tumorigenesis. Growth and progression of the transition-zone tumor are fueled by a combination of polyploid mitosis, endoreplication, and depolyploidization. Both polyploid mitosis and depolyploidization are error prone, resulting in chromosomal copy-number variation and polyaneuploidy. Comparative RNA-seq and epistasis analysis reveal that the DNA-damage response genes, also active during meiosis, are upregulated in these tumors and are required for the ploidy-reduction division. Together, these findings suggest that polyploidy and associated cell-cycle variants are critical for increased tumor-cell heterogeneity and genome instability during cancer progression.

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector.

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an autoinjector apparatus, trained operators can achieve more efficient and consistent transplantation results compared to those obtained using a manual injector. Here, we cover topics in a chronological fashion: from the crossing of Drosophila lines, to the induction and dissection of the primary tumor, transplantation of the primary tumor into a new adult host and continued generational transplantation of the tumor for extended studies. As a demonstration, here we use Notch intracellular domain (NICD) overexpression induced salivary gland imaginal ring tumors for generational transplantation. These tumors can first be reliably induced in a transition-zone microenvironment within larval salivary gland imaginal rings, then allografted and cultured in vivo to study continued tumor growth, evolution, and metastasis. This allotransplantation method can be useful in potential drug screening programs, as well as for studying tumor-host interactions.

Surgical options for submucosal tumors near the esophagogastric junction: does size or location matter?

BACKGROUND: Submucosal tumors (SMTs) of different etiologies exist from esophagus to rectum. Esophagogastric junction (EGJ) is one of the known difficult locations for tumor resection. Although minimally invasive surgery (MIS) is a well-established approach for gastrointestinal surgery, there is no consensus that MIS for resection of SMTs around EGJ is superior to laparotomy. We tried to clarify the factors that determine the surgeons' choices between these two approaches. METHODS: From January 2002 to June 2016, 909 patients with SMTs underwent resection in our department. Among them, 119 patients (13%) had SMTs around EGJ were enrolled by retrospective review. The clinicopathological features and tumor-related parameters were reviewed and analyzed. RESULTS: The cohort was stratified into three groups according to the extent of gastrectomy and surgical approaches. The three groups are as following: major gastrectomy (n = 13), minor gastrectomy by laparotomy (n = 51), and minor gastrectomy with MIS (n = 55). The average tumor size was significantly larger in the major gastrectomy group than in the two minor gastrectomy groups; however, there was no difference between the two minor gastrectomy groups (5.33 cm, 4.07 cm, and 3.69 cm, respectively). The minor gastrectomy with MIS required least hospital stay and operation duration also. We re-stratify the two minor gastrectomy groups (n = 106) according to the orientation of SMTs around the EGJ into 4 zones. Most of SMTs located on the greater curvature side of the EGJ were resected with MIS (82% versus 18%), whereas SMTs in the other zones were resected more often by laparotomy (59% versus 41%). There was no surgical mortality within the cohort, while minor gastrectomy with MIS yielded least number of leakages among the three groups. CONCLUSIONS: For SMTs around the EGJ, larger tumors (diameter of more than 5 cm) are more likely to be resected with major gastrectomy. To resect SMTs around the EGJ in a wedge-like (minor gastrectomy) fashion, tumors located other than the greater curvature side were more often resected by laparotomy. However, MIS yielded acceptable safety and surgical outcomes compared to conventional laparotomy for SMTs around the EGJ of the same size.

Rapamycin and hydroxychloroquine combination alters macrophage polarization and sensitizes glioblastoma to immune checkpoint inhibitors.

INTRODUCTION: The failure of immune checkpoint inhibitor (ICPi) on glioblastoma (GBM) treatment underscores the need for improving therapeutic strategy. We aimed to change tumor associated macrophage (TAM) from M2 type (anti-inflammatory) to M1 (pro-inflammatory) type to increase the therapeutic response of ICPi. We proposed that combined rapamycin (R) and hydroxychloroquine (Q) preferentially induce M2 cells death, as fatty acid oxidation was their major source of energy. METHODS: Macrophage polarization was characterized on mice and human macrophage cell lines by specific cytokines stimulation with or without RQ treatment under single culture or co-culture with GBM cell lines. Tumor sizes were evaluated on subcutaneous and intracranial GL261 mice models with or without RQ, anti-PD1 mAb treatment. Tumor volumes assessed by MRI scan and proportions of tumor infiltrating immune cells analyzed by flow cytometry were compared. RESULTS: In vitro RQ treatment decreased the macrophages polarization of M2, increased the phagocytic ability, and increased the lipid droplets accumulation. RQ treatment decreased the expression levels of CD47 and SIRPα on tumor cells and macrophage cells in co-culture experiments. The combination of RQ and anti-PD1 treatment was synergistic in action. Enhanced the intra-tumoral M1/M2 ratio, the CD8/CD4 ratio in the intracranial GL261 tumor model after RQ treatment were evident. CONCLUSION: We provide a rationale for manipulating the macrophage phenotype and increased the therapeutic effect of ICPi. To re-educate and re-empower the TAM/microglia opens an interesting avenue for GBM treatment.

Oncogenic Notch Triggers Neoplastic Tumorigenesis in a Transition-Zone-like Tissue Microenvironment.

During the initial stages of tumorigenesis, the tissue microenvironment where the pro-tumor cells reside plays a crucial role in determining the fate of these cells. Transition zones, where two types of epithelial cells meet, are high-risk sites for carcinogenesis, but the underlying mechanism remains largely unclear. Here, we show that persistent upregulation of Notch signaling induces neoplastic tumorigenesis in a transition zone between the salivary gland imaginal ring cells and the giant cells in Drosophila larvae. In this region, local endogenous JAK-STAT and JNK signaling creates a tissue microenvironment that is susceptible to oncogenic-Notch-induced tumorigenesis, whereas the rest of the salivary gland imaginal ring is refractory to Notch-induced tumor transformation. JNK signaling activates a matrix metalloprotease (MMP1) to promote Notch-induced tumorigenesis at the transition zone. These findings illustrate the significance of local endogenous inflammatory signaling in primary tumor formation.

Ethylene signaling involves in seeds germination upon submergence and antioxidant response elicited confers submergence tolerance to rice seedlings.

BACKGROUND: Flooding has negative impact on agriculture. The plant hormone ethylene is involved in plant growth and stress responses, which are important role in tolerance and adaptation regulatory mechanisms during submergence stress. Ethylene signaling crosstalk with gibberellin signaling enhances tolerance in lowland rice (Flood Resistant 13A) through a quiescence strategy or in deepwater rice through an escape strategy when rice is submerged. Information regarding ethylene-mediated priming in submergence stress tolerance in rice is scant. Here, we used 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, to evaluate the response in submerged rice seedlings. RESULTS: The germination rate and mean germination times of rice seeds was higher in seedlings under submergence only when ethylene signaling was inhibited by supplemented with silver nitrate (AgNO3). Reduced leaf chlorophyll contents and induced senescence-associated genes in rice seedlings under submergence were relieved by pretreatment with an ethylene precursor. The ethylene-mediated priming by pretreatment with an ethylene precursor enhanced the survival rate and hydrogen peroxide (H2O2) and superoxide (O2-) anion accumulation and affected antioxidant response in rice seedlings. CONCLUSIONS: Pretreatment with an ethylene precursor leads to reactive oxygen species generation, which in turn triggered the antioxidant response system, thus improving the tolerance of rice seedlings to complete submergence stress. Thus, H2O2 signaling may contribute to ethylene-mediated priming to submergence stress tolerance in rice seedlings.

Inhibition of Notch signaling by the p105 and p180 subunits of Drosophila chromatin assembly factor 1 is required for follicle cell proliferation.

Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. Here, we report another side of CAF1, wherein the subunits CAF1-p105 and CAF1-p180 (also known as CAF1-105 and CAF1-180, respectively) inhibit expression of Notch target genes and show this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt, also known as Pebbled), leading to Cut downregulation and inhibition of follicle cell mitosis. Our studies further show Notch is functionally responsible for these phenotypes observed in both the CAF1-p105- and CAF1-p180-deficient follicle cells. Moreover, we reveal that CAF1-p105- and CAF1-p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative-feedback regulatory loop between Cut and Hnt underlying CAF1-p105 and CAF-p180 regulation, which is crucial for follicle cell differentiation. In conclusion, our studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner.

Pharmacologically upregulated carcinoembryonic antigen-expression enhances the cytolytic activity of genetically-modified chimeric antigen receptor NK-92MI against colorectal cancer cells.

BACKGROUND: The natural killer cell line, NK-92MI, is cytotoxic against various types of cancer. The aim of this study was to develop chimeric antigen receptor-modified (CAR) NK-92MI cells targeting carcinoembryonic antigen-expressing (CEA) tumours and increase killing efficacy by pharmacologically modifying CEA-expression. RESULT: We generated anti-CEA-CAR NK-92MI cells by retroviral vector transduction. This genetically-modified cell line recognised and lysed high CEA-expressing tumour cell lines (LS174T) at 47.54 ± 12.60% and moderate CEA-expressing tumour cell lines (WiDr) at 31.14 ± 16.92% at a 5:1 effector: target (E/T) ratio. The cell line did not lyse low CEA-expressing tumour cells (HCT116) as they did their parental cells (NK-92MI cells). The histone deacetylase-inhibitor (HDAC) sodium butyrate (NaB) and the methylation-inhibitor 5-azacytidine (5-AZA), as epigenetic modifiers, induced CEA-expression in HCT116 and WiDr cells. Although the IC50 of 5 fluorouracil (5-FU) increased, both cell lines showed collateral sensitivity to anti-CEA-CAR NK-92MI cells. The cytolytic function of anti-CEA-CAR NK-92MI cells was increased from 22.99 ± 2.04% of lysis background to 69.20 ± 11.92% after NaB treatment, and 69.70 ± 9.93% after 5-AZA treatment, at a 10:1 E/T ratio in HCT116 cells. The WiDr cells showed similar trend, from 22.99 ± 4.01% of lysis background to 70.69 ± 10.19% after NaB treatment, and 59.44 ± 10.92% after 5-AZA treatment, at a 10:1 E/T ratio. CONCLUSIONS: This data indicates that the effector-ability of anti-CEA-CAR NK-92MI increased in a CEA-dependent manner. The combination of epigenetic-modifiers like HDAC-inhibitors, methylation-inhibitors, and adoptive-transfer of ex vivo-expanded allogeneic-NK cells may be clinically applicable to patients with in 5-FU resistant condition.

Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells.

Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo. Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation.

The m6A pathway facilitates sex determination in Drosophila.

The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.

Systematic analysis reveals tumor-enhancing and -suppressing microRNAs in Drosophila epithelial tumors.

Despite their emergence as an important class of noncoding RNAs involved in cancer cell transformation, invasion, and migration, the precise role of microRNAs (miRNAs) in tumorigenesis remains elusive. To gain insights into how miRNAs contribute to primary tumor formation, we conducted an RNA sequencing (RNA-Seq) analysis of Drosophila wing disc epithelial tumors induced by knockdown of a neoplastic tumor-suppressor gene (nTSG) lethal giant larvae (lgl), combined with overexpression of an active form of oncogene Ras (RasV12 ), and identified 51 mature miRNAs that changed significantly in tumorous discs. Followed by in vivo tumor enhancer and suppressor screens in sensitized genetic backgrounds, we identified 10 tumor-enhancing (TE) miRNAs and 11 tumor-suppressing (TS) miRNAs that contributed to the nTSG defect-induced tumorigenesis. Among these, four TE and three TS miRNAs have human homologs. From this study, we also identified 29 miRNAs that individually had no obvious role in enhancing or alleviating tumorigenesis despite their changed expression levels in nTSG tumors. This systematic analysis, which includes both RNA-Seq and in vivo functional studies, helps to categorize miRNAs into different groups based on their expression profile and functional relevance in epithelial tumorigenesis, whereas the evolutionarily conserved TE and TS miRNAs provide potential therapeutic targets for epithelial tumor treatment.