Network of mutually repressive metastasis regulators can promote cell heterogeneity and metastatic transitions.

The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.

Factor-V HR2 haplotype and thromboembolic disease.

OBJECTIVES: The objective of this study is to evaluate the prevalence of HR2 polymorphism among patients with pulmonary embolism (PE) and healthy subjects. BACKGROUND: Polymorphism in the factor V gene named HR2 has been described as a possible risk factor for venous thromboembolism (VTE) development. Contradictive results on this association have been reported. METHODS: Eighty-five patients admitted for PE and 72 healthy subjects were included in the study. Thrombophilia screening using genetic tests for factor V Leiden (G1691A/Leiden and HR2 haplotype) and other genetic mutations were investigated. RESULTS: Of 85 patients with PE, 20 (23.53%) carried the HR2 haplotype. Further, a majority of the patients with HR2 haplotype had recurrent venous thrombosis or PE (15 out of 20 patients). The HR2 haplotype was detected in 6 (8.3%) out of 72 healthy subjects. Patients had significantly higher HR2 haplotype frequency than healthy controls (P = 0.001). HR2 carriers had a three-fold increase in risk of developing PE (OR = 3.38, 95% CI = 1.27-8.96, P = 0.011). After adjustment for other tested defects for thrombophilia, HR2 haplotype was associated with increased risk of thromboembolic events (OR = 3.05, 95% CI = 1.11-8.35, P = 0.03). However, after adjustment for sex and age, HR2 polymorphism was no longer associated with the risk of thromboembolic event (OR = 1.22, 95% CI = 0.34-4.38, P = 0.76). CONCLUSIONS: Our study does not support the notion that factor V HR2 haplotype might be a risk factor for thrombosis despite its high prevalence among patients with PE.

A self-defeating anabolic program leads to ß-cell apoptosis in endoplasmic reticulum stress-induced diabetes via regulation of amino acid flux.

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing ß-cells in type 2 diabetes mellitus. ß-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in ß-cells in vivo. Paradoxically, chronic ER stress in ß-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in ß-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

Next-generation sequencing reveals additional HLA class I and class II alleles associated with type 1 diabetes and age at onset.

Introduction: Type 1 diabetes is an autoimmune disease with an significant genetic component, played mainly by the HLA class II genes. Although evidence on the role of HLA class I genes in developing type 1 diabetes and its onset have emerged, current HLA screening is limited to determining DR3 and DR4 haplotypes. This study aimed to investigate the role of HLA genes on type 1 diabetes risk and age of onset by extensive typing. Methods: This study included 115 children and young adults with type 1 diabetes for whom typing of HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 genes was conducted using Next Generation Sequencing. Results: We observed that 13% of type 1 diabetes subjects had non-classical HLA haplotypes that predispose to diabetes. We also found that compared to type 1 diabetes subjects with classical HLA haplotypes, non-classical HLA subjects had a significantly higher frequency of HLA-B*39:06:02 (p-value=0.01) and HLA-C*07:02:01 (p-value=0.03) alleles, known to be involved in activating the immune response. Non-classical HLA subjects also presented peculiar clinical features compared to classical HLA subjects, such as multiple diabetic antibodies and the absence of other autoimmune diseases (i.e., coeliac disease and thyroiditis). We also observed that subjects with early onset had a higher frequency of DQ2/DQ8 genotype than late-onset individuals. Moreover, subjects with late-onset had a higher frequency of alleles HLA-B*27 (p-value=0.003), HLA-C*01:02:01 (p-value=0.027) and C*02:02:02 (p-value=0.01), known to be associated with increased protection against viral infections. Discussion: This study reveals a broader involvement of the HLA locus in the development and onset of type 1 diabetes, providing insights into new possible disease prevention and management strategies.

eIF2alpha phosphorylation tips the balance to apoptosis during osmotic stress.

Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2alpha, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2alpha phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2alpha phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2alpha promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2alpha phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2alpha phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2alpha phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2alpha phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1.

Use of DPB1 T-cell epitope algorithm among italian transplant centers: A survey on behalf of Associazione Italiana di Immunogenetica e Biologia dei Trapianti.

The HLA-DPB1 locus has been demonstrated to have a significant role on patients' outcome after allogeneic HSCT, and the so-called T-cell epitope (TCE) algorithm has been incorporated in international guidelines for the selection of unrelated donors. The purpose of the present study is to measure, through a national survey conducted on behalf of the Associazione Italiana di Immunogenetica e Biologia dei Trapianti (AIBT), the extent of awareness and use of HLA-DPB1 TCE-based algorithms during the donor search. 89% of the HLA laboratories answered to a short questionnaire and the results showed a progressive increase of the laboratories typing DPB1 in patients and their potential donors during the search (from 44% to 79% during the 2010-2019 period) as well as the application of a TCE-based algorithm for the donor choice whenever possible (from 24% to 65% during the same period). The DP-permissiveness status is detailed in the official HLA typing report by 12%, 32% and 50% of laboratories in 2010, 2015 and 2019, respectively. The present data indicate an encouraging raise in the awareness of the HLA-DPB1 role in unrelated donor selection; noteworthy, mentioning the TCE-based permissiveness status in the HLA typing report of each potential unrelated donor represents a notable mean to raise awareness among transplant physicians and to support them in their task of choosing the best donor. Nonetheless, despite the compelling evidence of the predictive ability of TCE-based algorithms, further efforts are still needed to extend its application to all transplant centers in Italy.

Modeling the effect of 3 missense AGXT mutations on dimerization of the AGT enzyme in primary hyperoxaluria type 1.

INTRODUCTION: Mutations of the AGXT gene encoding the alanine:glyoxylate aminotransferase liver enzyme (AGT) cause primary hyperoxaluria type 1 (PH1). Here we report a molecular modeling study of selected missense AGXT mutations: the common Gly170Arg and the recently described Gly47Arg and Ser81Leu variants, predicted to be pathogenic using standard criteria. METHODS: Taking advantage of the refined 3D structure of AGT, we computed the dimerization energy of the wild-type and mutated proteins. RESULTS: Molecular modeling predicted that Gly47Arg affects dimerization with a similar effect to that shown previously for Gly170Arg through classical biochemical approaches. In contrast, no effect on dimerization was predicted for Ser81Leu. Therefore, this probably demonstrates pathogenic properties via a different mechanism, similar to that described for the adjacent Gly82Glu mutation that affects pyridoxine binding. CONCLUSION: This study shows that the molecular modeling approach can contribute to evaluating the pathogenicity of some missense variants that affect dimerization. However, in silico studies--aimed to assess the relationship between structural change and biological effects--require the integrated use of more than 1 tool.

HLA-DRB1 alleles and juvenile idiopathic arthritis: Diagnostic clues emerging from a meta-analysis.

Juvenile Idiopathic Arthritis (JIA) is characterized with a variable pattern of articular involvement and systemic symptoms and, thus, it has been classified in several subtypes. Genetic predisposition to JIA is mainly due to HLA class II molecules (HLA-DRB1, HLA-DPB1), although HLA class I molecules and non-HLA genes have been implicated, too. Here, we carried out a meta-analysis including selected studies designed to assess HLA genetic background of JIA patients, compared to healthy controls; particularly, we focused our attention on HLA-DRB1. In summary, our meta-analysis showed four main findings regarding HLA-DRB1 locus as a genetic factor of JIA: i) HLA-DRB1*08 is a strong factor predisposing to JIA, both for oligo-articular and poly-articular forms (oJIA>pJIA); ii) HLA-DRB1*01 and HLA-DRB1*04 may be involved in the genetic predisposition of Rheumatoid Factor (RF) positive forms of JIA; iii) HLA-DRB1*11 was confirmed to be predisposing to oligo-articular JIA; iv) HLA-DRB1*04 was confirmed to have a role in systemic JIA. Importantly, RF positivity seems to select the JIA clinical subset with the strongest immunogenetic similarities with adult rheumatoid arthritis.

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

SNAT2 silencing prevents the osmotic induction of transport system A and hinders cell recovery from hypertonic stress.

Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two siRNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress.

Warfarin-related nephropathy: possible role for the warfarin pharmacogenetic profile.

Warfarin-related nephropathy (WRN) is a renal complication of warfarin treatment associated with over-anticoagulation. We describe a case of a 73-year-old man affected by chronic kidney disease, essential hypertension and atrial fibrillation treated with warfarin. The patient presented a rapid course of kidney failure after many episodes of over-anticoagulation, and renal biopsy demonstrated WRN. Interestingly, the patient's warfarin pharmacogenetic profile showed that he was warfarin sensitive. This is the first report describing the presence of gene polymorphisms affecting warfarin metabolism in a subject with a biopsy-proven WRN. The patient was treated with corticosteroids obtaining a partial clinical response.

A prognostic gene signature for metastasis-free survival of triple negative breast cancer patients.

Although triple negative breast cancers (TNBC) are the most aggressive subtype of breast cancer, they currently lack targeted therapies. Because this classification still includes a heterogeneous collection of tumors, new tools to classify TNBCs are urgently required in order to improve our prognostic capability for high risk patients and predict response to therapy. We previously defined a gene expression signature, RKIP Pathway Metastasis Signature (RPMS), based upon a metastasis-suppressive signaling pathway initiated by Raf Kinase Inhibitory Protein (RKIP). We have now generated a new BACH1 Pathway Metastasis gene signature (BPMS) that utilizes targets of the metastasis regulator BACH1. Specifically, we substituted experimentally validated target genes to generate a new BACH1 metagene, developed an approach to optimize patient tumor stratification, and reduced the number of signature genes to 30. The BPMS significantly and selectively stratified metastasis-free survival in basal-like and, in particular, TNBC patients. In addition, the BPMS further stratified patients identified as having a good or poor prognosis by other signatures including the Mammaprint® and Oncotype® clinical tests. The BPMS is thus complementary to existing signatures and is a prognostic tool for high risk ER-HER2- patients. We also demonstrate the potential clinical applicability of the BPMS as a single sample predictor. Together, these results reveal the potential of this pathway-based BPMS gene signature to identify high risk TNBC patients that can respond effectively to targeted therapy, and highlight BPMS genes as novel drug targets for therapeutic development.

RKIP regulates MAP kinase signaling in cells with defective B-Raf activity.

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

Identification of novel metastasis suppressor signaling pathways for breast cancer.

Cancer lethality is mainly caused by metastasis. Therefore, understanding the nature of the genes involved in this process has become a priority. Given the heterogeneity of mutations in cancer cells, considerable focus has been directed toward characterizing metastasis genes in the context of relevant signaling pathways rather than treating genes as independent and equal entities. One signaling cascade implicated in the regulation of cell growth, invasion and metastasis is the MAP kinase pathway. Raf kinase inhibitory protein (RKIP) functions as an inhibitor of the MAP kinase pathway and is a metastasis suppressor in different cancer models. By utilizing statistical analysis of clinical data integrated with experimental validation, we recently identified components of the RKIP signaling pathway relevant to breast cancer metastasis. Using the RKIP pathway as an example, we show how prior biological knowledge can be efficiently combined with genome-wide patient data to identify gene regulatory mechanisms that control metastasis.

RKIP Suppresses Breast Cancer Metastasis to the Bone by Regulating Stroma-Associated Genes.

In the past decade cancer research has recognized the importance of tumorstroma interactions for the progression of primary tumors to an aggressive and invasive phenotype and for colonization of new organs in the context of metastasis. The dialogue between tumor cells and the surrounding stroma is a complex and dynamic phenomenon, as many cell types and soluble factors are involved. While the function of many of the players involved in this cross talk have been studied, the regulatory mechanisms and signaling pathways that control their expression haven't been investigated in depth. By using a novel, interdisciplinary approach applied to the mechanism of action of the metastasis suppressor, Raf kinase inhibitory protein (RKIP), we identified a signaling pathway that suppresses invasion and metastasis through regulation of stroma-associated genes. Conceptually, the approach we developed uses a master regulator and expression arrays from breast cancer patients to formulate hypotheses based on clinical data. Experimental validation is followed by further bioinformatic analysis to establish the clinical significance of discoveries. Using RKIP as an example we show here that this multi-step approach can be used to identify gene regulatory mechanisms that affect tumor-stroma interactions that in turn influence metastasis to the bone or other organs.

A novel interplay between Rap1 and PKA regulates induction of angiogenesis in prostate cancer.

Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.

Requirement of rRNA methylation for 80S ribosome assembly on a cohort of cellular internal ribosome entry sites.

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.

Genetic factors in mother-to-child transmission of HCV infection.

HCV infection transmission rate in infants born to HCV-positive mothers is about 5%. HIV co-infection and high maternal RNA viral load are associated with increased transmission. The only genetic factor previously evaluated is HLA. We investigated the role of genetic factors already associated in adults with HCV infection evolution (HLA-DRB1, MBL2, TNF-alpha, IFN-gamma and IL-10), or liver disease progression (HFE and TGF-beta1). 384 Italian subjects were recruited, including 38 HCV-positive mother/child pairs; 104 infected, non-transmitting mothers with their 114 children; 21 vertically infected children and 69 HCV-exposed, uninfected children. Samples were analysed for previously described gene polymorphisms. Maternal HLA-DRB104 correlated with protection from vertical transmission (p=0.023), while HLA-DRB110 in children was a risk factor (p=0.036). Investigation of concordance degree in HLA-DRB1 locus revealed that a HLA mismatch between mother and child was a protective factor (p=0.017) indicating that alloreactive immune responses are involved in preventing HCV vertical transmission.

The thioxotriazole copper(II) complex A0 induces endoplasmic reticulum stress and paraptotic death in human cancer cells.

The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2alpha phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2alpha, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2alpha phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2alpha phosphorylation and the UPR.

Differential control of the CCAAT/enhancer-binding protein beta (C/EBPbeta) products liver-enriched transcriptional activating protein (LAP) and liver-enriched transcriptional inhibitory protein (LIP) and the regulation of gene expression during the response to endoplasmic reticulum stress.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), is one of the genes with increased expression during ER stress. Translation of the C/EBPbeta mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPbeta-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2alpha. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPbeta in the ER-stress response program was demonstrated using C/EBPbeta-deficient mouse embryonic fibroblasts. It is shown that C/EBPbeta attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.