Opposing Functions Research Articles

Calcium Signaling Regulates Autophagy and Apoptosis.

Calcium (Ca2+) functions as a second messenger that is critical in regulating fundamental physiological functions such as cell growth/development, cell survival, neuronal development and/or the maintenance of cellular functions. The coordination among various proteins/pumps/Ca2+ channels and Ca2+ storage in various organelles is critical in maintaining cytosolic Ca2+ levels that provide the spatial resolution needed for cellular homeostasis. An important regulatory aspect of Ca2+ homeostasis is a store operated Ca2+ entry (SOCE) mechanism that is activated by the depletion of Ca2+ from internal ER stores and has gained much attention for influencing functions in both excitable and non-excitable cells. Ca2+ has been shown to regulate opposing functions such as autophagy, that promote cell survival; on the other hand, Ca2+ also regulates programmed cell death processes such as apoptosis. The functional significance of the TRP/Orai channels has been elaborately studied; however, information on how they can modulate opposing functions and modulate function in excitable and non-excitable cells is limited. Importantly, perturbations in SOCE have been implicated in a spectrum of pathological neurodegenerative conditions. The critical role of autophagy machinery in the pathogenesis of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, would presumably unveil avenues for plausible therapeutic interventions for these diseases. We thus review the role of SOCE-regulated Ca2+ signaling in modulating these diverse functions in stem cell, immune regulation and neuromodulation.

Neutrophils in cancer: heterogeneous and multifaceted.

Neutrophils are the most abundant myeloid cells in human blood and are emerging as important regulators of cancer. However, their functional importance has often been overlooked on the basis that they are short-lived, terminally differentiated and non-proliferative. Recent studies of their prominent roles in cancer have led to a paradigm shift in our appreciation of neutrophil functional diversity. This Review describes how neutrophil diversification, which in some contexts can lead to opposing functions, is generated within the tumour microenvironment as well as systemically. We compare neutrophil heterogeneity in cancer and in other pathophysiological contexts to provide an updated overview of our current knowledge of the functions of neutrophils in cancer.

Abstract LB210: MicroRNA function is malleable and can be governed by target gene expression levels

Abstract MicroRNAs are short, noncoding RNA molecules that reduce the expression of target genes by inhibiting messenger RNA translation and promoting messenger RNA degradation. They are highly dysregulated in cancer, with many microRNAs appearing to have potent oncogenic or tumor suppressive effects. As a result, there exists substantial interest in targeting microRNAs for cancer therapy. However, the literature is filled with conflicting accounts as to whether specific microRNAs are oncogenic or tumor suppressive, with many microRNAs appearing to exhibit entirely opposite functions when placed in different contexts. One explanation for this duality is that individual microRNAs can target tens to hundreds of different genes, many of which may possess opposing functions. Hence, it is likely that the expression levels of the target genes influence whether an individual microRNA will have a net oncogenic or net tumor suppressive effect. To examine this hypothesis, we developed a computational model of miR-125b's effect on the apoptosis pathway. This is an ideal system for examining the expression level hypothesis, as miR-125b is a microRNA that targets several closely interacting pro- and anti-apoptotic members of the pathway, which can be easily modeled as a system of differential equations. From the model, we elucidate a general trend that miR-125b is more pro-apoptotic when its anti-apoptotic targets are overexpressed and/or its pro-apoptotic targets are underexpressed, while it is more anti-apoptotic when its pro-apoptotic targets are overexpressed and/or its anti-apoptotic targets are underexpressed. To examine this experimentally, we switched miR-125b from being tumor suppressive to being oncogenic in cells by modifying the expression levels of one of miR-125b's anti-apoptotic targets. In addition, we observed that treating cells with anticancer drugs can also reverse miR-125b's status as a tumor suppressor. These results have important implications for the use of microRNAs as therapeutic targets in cancer. Citation Format: Alexander A. Svoronos, Stuart G. Campbell, Donald M. Engelman. MicroRNA function is malleable and can be governed by target gene expression levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB210.

Therapeutic implications of menin inhibition in acute leukemias.

Menin inhibitors are novel targeted agents currently in clinical development for the treatment of genetically defined subsets of acute leukemia. Menin has a tumor suppressor function in endocrine glands. Germline mutations in the gene encoding menin cause the multiple endocrine neoplasia type 1 (MEN1) syndrome, a hereditary condition associated with tumors of the endocrine glands. However, menin is also critical for leukemogenesis in subsets driven by rearrangement of the Lysine Methyltransferase 2A (KMT2A) gene, previously known as mixed-lineage leukemia (MLL), which encodes an epigenetic modifier. These seemingly opposing functions of menin can be explained by its various roles in gene regulation. Therefore, leukemias with rearrangement of KMT2A are predicted to respond to menin inhibition with early clinical data validating this proof-of-concept. These leukemias affect infants, children and adults, and lead to adverse outcomes with current standard therapies. Recent studies have identified novel targets in acute leukemia that are susceptible to menin inhibition, such as mutated Nucleophosmin 1 (NPM1), the most common genetic alteration in adult acute myeloid leukemia (AML). In addition to these alterations, other leukemia subsets with similar transcriptional dependency could be targeted through menin inhibition. This led to rationally designed clinical studies, investigating small-molecule oral menin inhibitors in relapsed acute leukemias with promising early results. Herein, we discuss the physiologic and malignant biology of menin, the mechanisms of leukemia in these susceptible subsets, and future therapeutic strategies using these inhibitors in acute leukemia.

Tumor restriction by type I collagen opposes tumor-promoting effects of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAF) may exert tumor-promoting and tumor-suppressive functions, but the mechanisms underlying these opposing effects remain elusive. Here, we sought to understand these potentially opposing functions by interrogating functional relationships among CAF subtypes, their mediators, desmoplasia, and tumor growth in a wide range of tumor types metastasizing to the liver, the most common organ site for metastasis. Depletion of hepatic stellate cells (HSC), which represented the main source of CAF in mice and patients in our study, or depletion of all CAF decreased tumor growth and mortality in desmoplastic colorectal and pancreatic metastasis but not in nondesmoplastic metastatic tumors. Single-cell RNA-Seq in conjunction with CellPhoneDB ligand-receptor analysis, as well as studies in immune cell-depleted and HSC-selective knockout mice, uncovered direct CAF-tumor interactions as a tumor-promoting mechanism, mediated by myofibroblastic CAF-secreted (myCAF-secreted) hyaluronan and inflammatory CAF-secreted (iCAF-secreted) HGF. These effects were opposed by myCAF-expressed type I collagen, which suppressed tumor growth by mechanically restraining tumor spread, overriding its own stiffness-induced mechanosignals. In summary, mechanical restriction by type I collagen opposes the overall tumor-promoting effects of CAF, thus providing a mechanistic explanation for their dual functions in cancer. Therapeutic targeting of tumor-promoting CAF mediators while preserving type I collagen may convert CAF from tumor promoting to tumor restricting.

Early Role of Rif1 in the Repair of P‐element Transposase‐Induced DNA Double Strand Breaks

Rap1 interacting factor (RIF1) is a structurally well-conserved protein in eukaryotes that plays an important role in pathway choice during the repair of DNA double strand breaks (DSBs). In human cells, Rif1 directly interacts with phosphorylated p53 binding protein (53BP1) to inhibit BRCA1 mediated resection of DSBs during G1, and hence promotes repair via non-homologous end joining. In contrast, Saccharomyces cerevisiae Rif1 was shown to promote resection and homology-directed repair of DSBs in both G1 and G2. Given the opposing repair functions of Rif1 in evolutionary distant mammalian and yeast cells, we have explored the DSB repair role of Rif1 in phylogenetic intermediate Drosophila melanogaster. Hatching assays and irradiation sensitivity assays were conducted to determine whether rif1 mutant fly stocks exhibit any DSB repair defect during embryonic development or larval development. No such defect was observed. Next, in order to probe whether Rif1 influences repair pathway choice, as is seen in mammals and yeast, DSB repair assays were conducted. These assays allow for the determination of repair mechanism—end-joining or homology-directed repair—of site-specific DSBs in the male germ-line. rif1 mutants exhibited no defects in the repair of I-SceI endonuclease-induced breaks but, interestingly, a near-complete defect in the repair of P-element transposase-induced breaks. These results suggest that Drosophila RIF1 plays an early role in the repair of P-element transposase-induced breaks and may provide insight into the disparate roles of RIF1 in yeast and mammals.

The E2F transcription factor 2: What do we know?

E2F transcription factor 2 (E2F2) is a member of the E2F family of transcription factors. The classical view is that some E2Fs act as "activators" and others "inhibitors" of cell cycle gene expression. However, the so-called "activator" E2F2 is particularly enigmatic, with seemingly contradictory roles in the cell cycle, proliferation, apoptosis, inflammation, and cell migration and invasion. How can we rationalize the apparently opposing functions of E2F2 in different situations? This is difficult because different methods of studying E2F2 have yielded conflicting results, so extrapolating mechanisms from an observed endpoint is challenging. This review will attempt to summarize and clarify these issues. This review focuses on genetic studies that have helped elucidate the biological functions of E2F2 and that have enhanced our understanding of how E2F2 is integrated into pathways controlling the cell cycle, proliferation, apoptosis, inflammation, and cell migration and invasion. This review will also discuss the function of E2F2 in cancer and other diseases. This review provides a strong basis for further research on the biological function and clinical potential of E2F2.

Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10.

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rβ, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rβ binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

Monosynaptic Retrograde Tracing From Prelimbic Neuron Subpopulations Projecting to Either Nucleus Accumbens Core or Rostromedial Tegmental Nucleus.

The prelimbic (PL) region of the medial prefrontal cortex (mPFC) has been implicated in both driving and suppressing motivated behaviors, including cocaine-seeking in rats. These seemingly opposing functions may be mediated by different efferent targets of PL projections, such as the nucleus accumbens (NAc) core and rostromedial tegmental nucleus (RMTg), which have contrasting roles in reward-seeking behaviors. We sought to characterize the anatomical connectivity differences between PL neurons projecting to NAc core and RMTg. We used conventional retrograde tracers to reveal distinct subpopulations of PL neurons projecting to NAc core vs. RMTg in rats, with very little overlap. To examine potential differences in input specificity for these two PL subpopulations, we then used Cre-dependent rabies virus (EnvA-RV-EGFP) as a monosynaptic retrograde tracer and targeted specific PL neurons via injections of retrograde CAV2-Cre in either NAc core or RMTg. We observed a similar catalog of cortical, thalamic, and limbic afferents for both NAc- and RMTg-projecting populations, with the primary source of afferent information arising from neighboring prefrontal neurons in ipsilateral PL and infralimbic cortex (IL). However, when the two subpopulations were directly compared, we found that RMTg-projecting PL neurons received a greater proportion of input from ipsilateral PL and IL, whereas NAc-projecting PL neurons received a greater proportion of input from most other cortical areas, mediodorsal thalamic nucleus, and several other subcortical areas. NAc-projecting PL neurons also received a greater proportion of contralateral cortical input. Our findings reveal that PL subpopulations differ not only in their efferent target but also in the input specificity from afferent structures. These differences in connectivity are likely to be critical to functional differences of PL subpopulations.

Highly efficient water desalination through hourglass shaped carbon nanopores

Biological nanopores such as aquaporins combine the opposing functions of high water permeation and total ion exclusion in part by the virtue of their hourglass shape. Here, we perform molecular dynamics simulations to examine water and ion conduction through hourglass shaped nanopores created from carbon nanotubes (CNTs) of chirality (6,6), (8,8), and (10,10) in combination with carbon nanocones of half cone angles 41.8°, 30.0°, 19.45°, 9.6° and 0.0°. We observe large variations in flow through the nanopores with change in half cone angles and tube diameters. By computing the pore-water interactions we find a correlated change between the flux and the density profiles of water inside the nanopores. Further, from the orientation, and the hydrogen bonding characteristics of water, we uncover some unexplored facets of flow through hourglass shaped nanopores. The results are insightful for devising novel separation membranes based on nanopores that mimic the shape of biological nanochannels.

The FasLane to ocular pathology-metalloproteinase cleavage of membrane-bound FasL determines FasL function.

Fas ligand (FasL) is best known for its ability to induce cell death in a wide range of Fas-expressing targets and to limit inflammation in immunoprivileged sites such as the eye. In addition, the ability of FasL to induce a much more extensive list of outcomes is being increasingly explored and accepted. These outcomes include the induction of proinflammatory cytokine production, T cell activation, and cell motility. However, the distinct and opposing functions of membrane-associated FasL (mFasL) and the C-terminal soluble FasL fragment (sFasL) released by metalloproteinase cleavage is less well documented and understood. Both mFasL and sFasL can form trimers that engage the trimeric Fas receptor, but only mFasL can form a multimeric complex in lipid rafts to trigger apoptosis and inflammation. By contrast, a number of reports have now documented the anti-apoptotic and anti-inflammatory activity of sFasL, pointing to a critical regulatory function of the soluble molecule. The immunomodulatory activity of FasL is particularly evident in ocular pathology where elimination of the metalloproteinase cleavage site and the ensuing increased expression of mFasL can severely exacerbate the extent of inflammation and cell death. By contrast, both homeostatic and increased expression of sFasL can limit inflammation and cell death. The mechanism(s) responsible for the protective activity of sFasL are discussed but remain controversial. Nevertheless, it will be important to consider therapeutic applications of sFasL for the treatment of ocular diseases such as glaucoma.

C-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 can act as either tumor suppressors or pro-oncogenic kinases in human cancers. The isoform-specific roles for JNK1 and JNK2 in human pancreatic cancer are still unclear, the question which should be addressed in this project. Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 clones were established either expressing either JNK1 or -2 shRNA in a stable manner. Basal anchorage-dependent and –independent cell growth, single-cell movement, and invasion using the Boyden chamber assay were analyzed. Xenograft growth was assessed using an orthotopic mouse model. All seven tested pancreatic cancer cell lines expressed JNKs as did human pancreatic cancer samples determined by immunohistochemistry. Pharmacological, unspecific JNK inhibition (SP600125) reduced cell growth of all cell lines but PANC-1. Especially inhibition of JNK2 resulted in overall increased oncogenic potential with increased proliferation and invasion, associated with alterations in cytoskeleton structure. Specific inhibition of JNK1 revealed opposing functions. Overall, JNK1 and JNK2 can exert different functions in human pancreatic cancer and act as counter players for tumor invasion. Specifically modulating the activity of JNKs may be of potential therapeutic interest in the future.

The curious case of miR-155 in SLE.

Epigenetic modifications have been well documented in autoimmune diseases. MicroRNAs (miRNAs), in particular, have long intrigued scientists in the field of autoimmunity. Owing to its central role in the development of the immune system, microRNA-155 (miR-155) is deeply involved in systemic lupus erythematosus (SLE). Despite the advancements made in treating SLE, the disease still remains incurable. Therefore, recent attention has been drawn to the manipulation of epigenetics in the development of curative treatments. In fact, it is a widely held view that miRNA-targeted therapy is a new glimmer of hope in the treatment of autoimmune diseases. However, the duplicity of miRNAs should not be overlooked. A single miRNA can target several mRNAs, and some mRNAs may possess opposing functions. In this review, we highlight the role of miR-155 as a biomarker and review its functions in SLE patients and animal models while discussing possible reasons behind inconsistencies across studies.

The p75NTR and its carboxyl-terminal fragment exert opposing effects on melanoma cell proliferation and apoptosis via modulation of the NF-κB pathway.

The p75 neurotrophin receptor (p75NTR ), a member of the tumor necrosis factor superfamily of receptors, is sensitive to proteolysis and has been observed to be expressed in various cancers. However, the roles of p75NTR and its proteolytic fragments in tumorigenesis remain incompletely understood. Here, we report that the proportion of the p75NTR carboxyl-terminal fragment (p75NTR -CTF) is much higher than that of the full-length p75NTR (p75NTR -FL) in melanoma cells. Whereas p75NTR -FL positively regulates apoptosis, p75NTR -CTF promotes cell proliferation and survival, as well as increasing sorafenib resistance invivo and invitro. Moreover, p75NTR -CTF activates the nuclear factor kappa B pathway and enhances the mRNA and protein levels of its downstream genes c-IAP1/2, FLIP, bFGF, IL8 and VEGF. On the contrary, p75NTR -FL inhibits these processes. Taken together, these findings demonstrate that p75NTR -CTF and p75NTR -FL have opposing functions in melanoma cells, suggesting that the ratio of the two proteins affects the balance between cell death and survival. The presence of distinct p75NTR proteolytic fragments may affect biological outcomes in tumor cells.

The Role of β-Arrestins in Regulating Stem Cell Phenotypes in Normal and Tumorigenic Cells.

β-Arrestins (ARRBs) are ubiquitously expressed scaffold proteins that mediate inactivation of G-protein-coupled receptor signaling, and in certain circumstances, G-protein independent pathways. Intriguingly, the two known ARRBs, β-arrestin1 (ARRB1) and β-Arrestin2 (ARRB2), seem to have opposing functions in regulating signaling cascades in several models in health and disease. Recent evidence suggests that ARRBs are implicated in regulating stem cell maintenance; however, their role, although crucial, is complex, and there is no universal model for ARRB-mediated regulation of stem cell characteristics. For the first time, this review compiles information on the function of ARRBs in stem cell biology and will discuss the role of ARRBs in regulating cell signaling pathways implicated in stem cell maintenance in normal and malignant stem cell populations. Although promising targets for cancer therapy, the ubiquitous nature of ARRBs and the plethora of functions in normal cell biology brings challenges for treatment selectivity. However, recent studies show promising evidence for specifically targeting ARRBs in myeloproliferative neoplasms.

An AKT2-specific nanobody that targets the hydrophobic motif induces cell cycle arrest, autophagy and loss of focal adhesions in MDA-MB-231 cells

The AKT kinase family is a high-profile target for cancer therapy. Despite their high degree of homology the three AKT isoforms (AKT1, AKT2 and AKT3) are non-redundant and can even have opposing functions. Small-molecule AKT inhibitors affect all three isoforms which severely limits their usefulness as research tool or therapeutic. Using AKT2-specific nanobodies we examined the function of endogenous AKT2 in breast cancer cells. Two AKT2 nanobodies (Nb8 and Nb9) modulate AKT2 and reduce MDA-MB-231 cell viability/proliferation. Nb8 binds the AKT2 hydrophobic motif and reduces IGF-1-induced phosphorylation of this site. This nanobody also affects the phosphorylation and/or expression levels of a wide range of proteins downstream of AKT, resulting in a G0/G1 cell cycle arrest, the induction of autophagy, a reduction in focal adhesion count and loss of stress fibers. While cell cycle progression is likely to be regulated by more than one isoform, our results indicate that both the effects on autophagy and the cytoskeleton are specific to AKT2. By using an isoform-specific nanobody we were able to map a part of the AKT2 pathway. Our results confirm AKT2 and the hydrophobic motif as targets for cancer therapy. Nb8 can be used as a research tool to study AKT2 signalling events and aid in the design of an AKT2-specific inhibitor.

Opposing Roles of Type I Interferons in Cancer Immunity.

The immune system is tasked with identifying malignant cells to eliminate or prevent cancer spread. This involves a complex orchestration of many immune cell types that together recognize different aspects of tumor transformation and growth. In response, tumors have developed mechanisms to circumvent immune attack. Type I interferons (IFN-Is) are a class of proinflammatory cytokines produced in response to viruses and other environmental stressors. IFN-Is are also emerging as essential drivers of antitumor immunity, potently stimulating the ability of immune cells to eliminate tumor cells. However, a more complicated role for IFN-Is has arisen, as prolonged stimulation can promote feedback inhibitory mechanisms that contribute to immune exhaustion and other deleterious effects that directly or indirectly permit cancer cells to escape immune clearance. We review the fundamental and opposing functions of IFN-Is that modulate tumor growth and impact immune function and ultimately how these functions can be harnessed for the design of new cancer therapies.

Ancient Cytokine Interleukin 15-Like (IL-15L) Induces a Type 2 Immune Response

Related interleukin-2, -15, and -15-like (IL-2, -15, and -15L) are ancient cytokines, with all three genes surviving in extant fish and some mammals. The present study is the first to identify IL-15L functions, namely in rainbow trout. In isolated trout splenocytes, and in vivo, purified recombinant IL-15L+IL-15Rα molecules induced expression of IL-4 and IL-13 homologs, which are markers of type 2 immunity. In contrast, trout IL-15 stimulated type 1 immunity markers, thus IL-15 and IL-15L can have opposing functions. Trout IL-15L was more dependent on “in trans” presentation by the receptor chain IL-15Rα than IL-15, and stimulated CD4−CD8−(IgM−) lymphocytes from thymus and spleen. We propose an important role for IL-15L early in the type 2 immunity cytokine cascade. Trout IL-2 and IL-15 exhibited features reminiscent of their mechanistic and functional dichotomy observed in mammals; for example, IL-15 but not IL-2 required a receptor alpha chain (only IL-15Rα in the case of fish) for its stability, and only IL-15 was efficient in stimulating lymphocytes from mucosal tissues. Data suggest that IL-15L and IL-15 may be particularly effective in stimulating innate lymphocyte type 2 cells (ILC2) and natural killer (NK) cells, respectively, but further identification of the cell types is needed. An interesting finding different from in mammals was the efficient stimulation of CD4+CD8+ thymocytes by IL-2. In short, this study presents fundamental information on the evolution of the IL-2/15/15L cytokine family.

P50 suppresses cytotoxic T lymphocyte effector function to regulate tumor immune escape and response to immunotherapy

BackgroundNF-κB is a key link between inflammation and cancer. Previous studies of NF-κB have largely focused on tumor cells, and the intrinsic function of NF-κB in T cells in tumor...

Potential Role of Gene Regulator NFAT5 in the Pathogenesis of Diabetes Mellitus.

Nuclear factor of activated T cells 5 (NFAT5), a Rel/nuclear factor- (NF-) κB family member, is the only known gene regulator of the mammalian adaptive response to osmotic stress. Exposure to elevated glucose increases the expression and nuclear translocation of NFAT5, as well as NFAT5-driven transcriptional activity in vivo and in vitro. Increased expression of NFAT5 is closely correlated with the progression of diabetes in patients. The distinct structure of NFAT5 governs its physiological and pathogenic roles, indicating its opposing functions. The ability of NFAT5 to maintain cell homeostasis and proliferation is impaired in patients with diabetes. NFAT5 promotes the formation of aldose reductase, pathogenesis of diabetic vascular complications, and insulin resistance. Additionally, NFAT5 activates inflammation at a very early stage of diabetes and induces persistent inflammation. Recent studies revealed that NFAT5 is an effective therapeutic target for diabetes. Here, we describe the current knowledge about NFAT5 and its relationship with diabetes, focusing on its diverse regulatory functions, and highlight the importance of this protein as a potential therapeutic target in patients with diabetes.