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.

Polyploidy in development and tumor models in Drosophila.

Polyploidy, a cell status defined as more than two sets of genomic DNA, is a conserved strategy across species that can increase cell size and biosynthetic production, but the functional aspects of polyploidy are nuanced and vary across cell types. Throughout Drosophila developmental stages (embryo, larva, pupa and adult), polyploid cells are present in numerous organs and help orchestrate development while contributing to normal growth, well-being and homeostasis of the organism. Conversely, increasing evidence has shown that polyploid cells are prevalent in Drosophila tumors and play important roles in tumor growth and invasiveness. Here, we summarize the genes and pathways involved in polyploidy during normal and tumorigenic development, the mechanisms underlying polyploidization, and the functional aspects of polyploidy in development, homeostasis and tumorigenesis in the Drosophila model.

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.

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.

Evolutionarily Conserved Roles for Apontic in Induction and Subsequent Decline of Cyclin E Expression.

Cyclin E is a key factor for S phase entry, and deregulation of Cyclin E results in developmental defects and tumors. Therefore, proper cycling of Cyclin E is crucial for normal growth. Here we found that transcription factors Apontic (Apt) and E2f1 cooperate to induce cyclin E in Drosophila. Functional binding motifs of Apt and E2f1 are clustered in the first intron of Drosophila cyclin E and directly contribute to the cyclin E transcription. Knockout of apt and e2f1 together abolished Cyclin E expression. Furthermore, Apt up-regulates Retinoblastoma family protein 1 (Rbf1) for proper chromatin compaction, which is known to repress cyclin E. Notably, Apt-dependent up-regulation of Cyclin E and Rbf1 is evolutionarily conserved in mammalian cells. Our findings reveal a unique mechanism underlying the induction and subsequent decline of Cyclin E expression.

Apontic directly activates hedgehog and cyclin E for proper organ growth and patterning.

Hedgehog (Hh) signaling pathway and Cyclin E are key players in cell proliferation and organ development. Hyperactivation of hh and cyclin E has been linked to several types of cancer. However, coordination of the expression of hh and cyclin E was not well understood. Here we show that an evolutionarily conserved transcription factor Apontic (Apt) directly activates hh and cyclin E through its binding site in the promoter regions of hh and cyclin E. This Apt-dependent proper expression of hh and cyclin E is required for cell proliferation and development of the Drosophila wing. Furthermore, Fibrinogen silencer-binding protein (FSBP), a mammalian homolog of Apt, also positively regulates Sonic hh (Shh), Desert hh (Dhh), Cyclin E1 (CCNE1) and Cyclin E2 (CCNE2) in cultured human cells, suggesting evolutionary conservation of the mechanism. Apt-mediated expression of hh and cyclin E can direct proliferation of Hh-expressing cells and simultaneous growth, patterning and differentiation of Hh-recipient cells. The discovery of the simultaneous expression of Hh and principal cell-cycle regulator Cyclin E by Apt implicates insight into the mechanism by which deregulated hh and cyclin E promotes tumor formation.

The T-box transcription factor Midline regulates wing development by repressing wingless and hedgehog in Drosophila.

Wingless (Wg) and Hedgehog (Hh) signaling pathways are key players in animal development. However, regulation of the expression of wg and hh are not well understood. Here, we show that Midline (Mid), an evolutionarily conserved transcription factor, expresses in the wing disc of Drosophila and plays a vital role in wing development. Loss or knock down of mid in the wing disc induced hyper-expression of wingless (wg) and yielded cocked and non-flat wings. Over-expression of mid in the wing disc markedly repressed the expression of wg, DE-Cadherin (DE-Cad) and armadillo (arm), and resulted in a small and blistered wing. In addition, a reduction in the dose of mid enhanced phenotypes of a gain-of-function mutant of hedgehog (hh). We also observed repression of hh upon overexpression of mid in the wing disc. Taken together, we propose that Mid regulates wing development by repressing wg and hh in Drosophila.

Therapeutic effects of PADRE-BAFF autovaccine on rat adjuvant arthritis.

B cell activating factor (BAFF) is a cytokine of tumor necrosis factor family mainly produced by monocytes and dendritic cells. BAFF can regulate the proliferation, differentiation, and survival of B lymphocytes by binding with BAFF-R on B cell membrane. Accumulating evidences showed that BAFF played crucial roles and was overexpressed in various autoimmune diseases such as systemic lupus erythematous (SLE) and rheumatoid arthritis (RA). This suggests that BAFF may be a therapeutic target for these diseases. In the present study, we developed a BAFF therapeutic vaccine by coupling a T helper cell epitope AKFVAAWTLKAA (PADRE) to the N terminus of BAFF extracellular domains (PADRE-BAFF) and expressed this fusion protein in Escherichia coli. The purified vaccine can induce high titer of neutralizing BAFF antibodies and ameliorate the syndrome of complete Freund's adjuvant (CFA) induced rheumatoid arthritis in rats. Our data indicated that the BAFF autovaccine may be a useful candidate for the treatment of some autoimmune diseases associated with high level of BAFF.

The role of indoleamine 2,3-dioxygenase (IDO) in immune tolerance: focus on macrophage polarization of THP-1 cells.

Macrophages can be divided into two groups as M1 and M2 phenotype. Our results and other groups revealed that IFN-γ can up-regulate the IDO expression and differentiate THP-1 cells to M1 phenotype. Therefore we hypothesized that IDO may play potential roles in macrophage differentiation. Interesting, our results indicated that the ectopic IDO increases the expression of M2 markers such as IL-10 and CXCR4 while decreases the M1 markers such as CCR7 and IL-12p35. In contrast, the knockdown of IDO expression in THP-1 cells resulted in increased M1 markers and lower M2 markers. Our results suggested that the expression intensity of IDO modulates macrophages differentiation. These finding support the counter-regulatory role for IDO with regarding to the polarization of macrophages to restrain excessive or inappropriate immune activation in inflammatory or tumor microenvironment. It throws new light on the mechanisms about the immunosuppressive effect of IDO in tumor or inflammatory diseases.

Nodal promotes the generation of M2-like macrophages and downregulates the expression of IL-12.

Nodal, a member of the TGF-ß superfamily, is an embryonic morphogen that is upregulated in different types of tumors. Nodal increases the tumorigenesis by inducing angiogenesis and promoting metastasis. Importantly, Nodal inhibition suppresses the growth and invasion of tumor. Since tumor-associated macrophages (TAMs) are the major infiltrating leukocytes in most cancers, we investigated whether Nodal is involved in the differentiation of TAMs. Our results revealed that Nodal inhibition in tumor microenvironment upregulated the production of IL-12 in macrophages and reversed TAMs to classically activated macrophage phenotype. In contrast, treatment with recombinant Nodal (rNodal) decreased the expression of IL-12 in murine macrophages. Furthermore, rNodal promoted macrophage polarization to an alternatively activated macrophage-like/TAM phenotype and modulated its function. These results suggest that Nodal may play an important role in macrophage polarization and downregulation of IL-12. The rescued antitumor function of TAMs via the inhibition of Nodal expression could be a new therapeutic strategy for cancer treatment.

Stabilization of Snail through AKT/GSK-3ß signaling pathway is required for TNF-α-induced epithelial-mesenchymal transition in prostate cancer PC3 cells.

Metastasis induced by chronic inflammation has been considered as a major challenge during cancer therapy. Epithelial-mesenchymal transition (EMT) is associated with cancer invasion and metastasis promoted by pro-inflammatory cytokine TNFα. However, the mechanisms underlying TNFα-induced EMT in prostate cancer cells is not entirely clear. Here we showed that EMT induced by longstanding stimulation with TNFα in prostate cancer PC3 cells is mediated by up-regulation of the transcriptional repressor Snail. TNFα-mediated EMT was characterized by acquiring mesenchymal fusiform morphology, increasing the expression of Vimentin and decreasing the expression of E-cadherin. Exposure to TNFα increased the expression of transcription factor Snail via post-transcriptional regulation process and induced Snail nuclear localization in PC3 cells. Moreover, overexpressed Snail in PC3 cells induced EMT. Conversely, suppressing Snail expression abrogated TNFα-induced EMT, suggesting that Snail plays a crucial role in TNFα-induced EMT in prostate cancer cells. Finally, we showed that TNFα time-dependently activated NF-κB, AKT, ERK, p38 MAPK signaling pathways, and elevated Snail stability by activating AKT pathway that subsequently inhibited GSK-3ß activity. Taken together, these results reveal that stabilization of Snail via AKT/GSK-3ß signaling pathway is required for TNFα-induced EMT in prostate cancer cells. This study offers a better understanding of TNFα-induced metastasis and provides an effective therapeutic strategy for prostate cancer treatment.

Epithelial-mesenchymal transition (EMT) induced by TNF-α requires AKT/GSK-3ß-mediated stabilization of snail in colorectal cancer.

Chronic inflammation-promoted metastasis has been considered as a major challenge in cancer therapy. Pro-inflammatory cytokine TNFα can induce cancer invasion and metastasis associated with epithelial-mesenchymal transition (EMT). However, the underlying mechanisms are not entirely clear. In this study, we showed that TNFα induces EMT in human HCT116 cells and thereby promotes colorectal cancer (CRC) invasion and metastasis. TNFα-induced EMT was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin and fibronectin with a concomitant decrease of E-cadherin and Zona occludin-1(ZO-1). TNFα treatment also increased the expression of transcription factor Snail, but not Slug, ZEB1 and Twist. Overexpression of Snail induced a switch from E-cadherin to N-cadherin expression in HCT116 cells, which is a characteristic of EMT. Conversely, knockdown of Snail significantly attenuated TNFα-induced EMT in HCT116 cells, suggesting that Snail plays a crucial role in TNFα-induced EMT. Interestingly, exposure to TNFα rapidly increased Snail protein expression and Snail nuclear localization but not mRNA level upregulation. Finally, we demonstrated that TNFα elevated Snail stability by activating AKT pathway and subsequently repressing GSK-3ß activity and decreasing the association of Snail with GSK-3ß. Knockdown of GSK-3ß further verified our finding. Taken together, these results revealed that AKT/GSK-3ß-mediated stabilization of Snail is required for TNFα-induced EMT in CRC cells. Our study provides a better understanding of inflammation-induced CRC metastasis.

T63, a new 4-arylidene curcumin analogue, induces cell cycle arrest and apoptosis through activation of the reactive oxygen species-FOXO3a pathway in lung cancer cells.

Curcumin (diferuloylmethane) is a natural polyphenol product of the plant Curcuma longa and has a diversity of antitumor activities. T63, a new 4-arylidene curcumin analogue, was reported to inhibit proliferation of lung cancer cells. However, its precise molecular antitumor mechanisms have not been well elucidated. Here, we showed that T63 could significantly inhibit the proliferation of A549 and H460 human lung cell lines via induction of G0/G1 cell cycle arrest and apoptosis. We found that the reactive oxygen species (ROS)-activated FOXO3a cascade plays a central role in T63-induced cell proliferation inhibition. Mechanistically, enhancement of ROS production by T63 induced FOXO3a expression and nuclear translocation through activation of p38MAPK and inhibition of AKT, subsequently elevating the expression of FOXO3a target genes, including p21, p27, and Bim, and then increased the levels of activated caspase-3 and decreased the levels of cyclin D1. Moreover, the antioxidant N-acetylcysteine markedly blocked the above effects, and small interfering RNA-mediated knockdown of FOXO3a also significantly decreased T63-induced cell cycle arrest and apoptosis. In vivo experiments showed that T63 significantly suppressed the growth of A549 lung cancer xenograft tumors, associated with proliferation suppression and apoptosis induction in tumor tissues, without inducing any notable major organ-related toxicity. These data indicated that the novel curcumin analogue T63 is a potent antitumor agent that induces cell cycle arrest and apoptosis and has significant therapeutic potential for lung cancer.

Resistin does not down-regulate the transcription of insulin receptor promoter.

OBJECTIVE: To detect the effect of resistin on the transcription of insulin receptor promoter. METHODS: Luciferase reporter gene was fused downstream of human insulin receptor promoter and the enzymatic activity of luciferase was determined in the presence or absence of resistin. The resistin expressed with plasmid was stained with antibody against Myc tag which was in frame fused with resistin coding sequence, and then imaged with confocal microscopy. RESULTS: The treatment of pIRP-LUC transfected cells with recombinant resistin did not result in significant difference in the enzymatic activity of luciferase compared to the untreated cells. Cell staining showed that green fluorescence could be observed in the cytoplasm, but not in the nucleus. CONCLUSION: The results suggest that the endogenous resistin may functionally locate in the cytoplasm, but does not enter the nucleus and not down-regulate the transcription of insulin receptor promoter.

[Assembly of full-length human resistin gene in vitro and construction of its eukaryotic expression vector].

OBJECTIVE: To assemble the full-length of human resistin gene in vitro by using oligonucleotides and to construct its eukaryotic expression vector. METHODS: According to the gene sequence of resistin (GenBank: AF323081), 10 oligonucleotides were designed and synthesized, followed by a touch down PCR to assemble the full-length gene. The PCR products were cloned into pSecTag2B vector and confirmed by sequencing. RESULTS: The band of PCR products and gene sequencing showed the insert fragment in pSecTag2B vector was identical to that as designed. CONCLUSION: The full-length of human resistin coding sequence was successfully assembled and amplified by touch down PCR, and a resistin-expressing eukaryotic vector was constructed.