Risk and survival in colorectal cancer with increasing body mass index: A nationwide population-based cohort study.

AIM: The aim was to explore potential associations between the body mass index (BMI) and the risk of colorectal cancer (CRC), including subsites of the colon, and cancer-specific death. METHODS: A registry-based cohort study was conducted with baseline data gathered from the Norwegian Tuberculosis Screening Programme, collected between 1963 and 1975, and linked to follow-up data from the Cancer Registry of Norway and the Norwegian Cause of Death Registry. Cox regression models were used to explore associations between BMI and CRC risk and cancer-specific death. RESULTS: Of 1 723 692 included individuals, 76 616 developed CRC during 55 370 707 person-years of follow-up. In men, a 5 kg/m2 increase in BMI was associated with an increased risk of colon cancer, including both right and left subsites, and rectal cancer. Allowing for nonlinearities, we found a U-shaped association for the right colon and an inverse U-shape for the left colon and rectum cancer. In women, a 5 kg/m2 increase in BMI in early adulthood was associated with increased risk of colon cancer, including both subsites. In women, an increased risk of CRC death with increasing BMI was found for colon cancer. CONCLUSIONS: Men of all ages have an increased risk of CRC with increasing BMI, with the highest risk for right-sided colon cancer. An increased risk for colon cancer was also found in women with high BMI in early adulthood. Furthermore, women of all age groups appeared to have an increased risk of CRC death with higher BMI.

Body mass index and pancreatic adenocarcinoma: A nationwide registry-based cohort study.

BACKGROUND AND OBJECTIVE: An association between body mass index (BMI) and pancreatic cancer is suggested in observational studies. However, further studies are required to substantiate available evidence. The aim of this study was to explore the association between BMI and pancreatic ductal adenocarcinoma (PDAC) risk, treatment, and mortality. METHODS: A registry-based cohort study was performed by combining data from four registries in Norway. Baseline data were collected between 1963 and 1975 with follow-up data collected until 2018. Kaplan-Meier curves and multivariable Cox regressions were estimated. Chi-square tests were used to analyze differences between groups. RESULTS: The study cohort consisted of 1,723,692 individuals. A total of 8973 PDAC cases were identified during 55,744,749 person-years of follow-up. A 5 kg/m2 increase in BMI was associated with an increased risk of PDAC if high BMI at young age (16-29 years) (hazard ratio (HR): 1.21, 95% confidence interval (CI): 1.13-1.31), both for men (HR: 1.30, 95% CI: 1.15-1.46) and women (HR: 1.16, 95% CI: 1.05-1.28). In men, there was a 52% increase in risk of early-onset PDAC (

Proteome Analysis of Pancreatic Tumors Implicates Extracellular Matrix in Patient Outcome.

Pancreatic cancer remains a disease with unmet clinical needs and inadequate diagnostic, prognostic, and predictive biomarkers. In-depth characterization of the disease proteome is limited. This study thus aims to define and describe protein networks underlying pancreatic cancer and identify protein centric subtypes with clinical relevance. Mass spectrometry-based proteomics was used to identify and quantify the proteome in tumor tissue, tumor-adjacent tissue, and patient-derived xenografts (PDX)-derived cell lines from patients with pancreatic cancer, and tissues from patients with chronic pancreatitis. We identified, quantified, and characterized 11,634 proteins from 72 pancreatic tissue samples. Network focused analysis of the proteomics data led to identification of a tumor epithelium-specific module and an extracellular matrix (ECM)-associated module that discriminated pancreatic tumor tissue from both tumor adjacent tissue and pancreatitis tissue. On the basis of the ECM module, we defined an ECM-high and an ECM-low subgroup, where the ECM-high subgroup was associated with poor prognosis (median survival months: 15.3 vs. 22.9 months; log-rank test, P = 0.02). The ECM-high tumors were characterized by elevated epithelial-mesenchymal transition and glycolytic activities, and low oxidative phosphorylation, E2F, and DNA repair pathway activities. This study offers novel insights into the protein network underlying pancreatic cancer opening up for proteome precision medicine development. Significance: Pancreatic cancer lacks reliable biomarkers for prognostication and treatment of patients. We analyzed the proteome of pancreatic tumors, nonmalignant tissues of the pancreas and PDX-derived cell lines, and identified proteins that discriminate between patients with good and poor survival. The proteomics data also unraveled potential novel drug targets.

cAMP-Mediated Autophagy Promotes Cell Survival via ROS-Induced Activation of PARP1: Implications for Treatment of Acute Lymphoblastic Leukemia.

DNA-damaging therapy is the basis for treatment of most cancers, including B-cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL). We have previously shown that cAMP-activating factors present in the bone marrow render ALL cells less sensitive to DNA damage-induced apoptosis, by enhancing autophagy and suppressing p53. To sensitize ALL cells to DNA-damaging therapy, we have searched for novel targets that may counteract the effects induced by cAMP signaling. In the current study, we have identified PARP1 as a potential target. We show that the PARP1 inhibitors olaparib or PJ34 inhibit cAMP-mediated autophagy and thereby potentiate the DNA-damaging treatment. Furthermore, we reveal that cAMP-mediated PARP1 activation is preceded by induction of reactive oxygen species (ROS) and results in depletion of nicotinamide adenine dinucleotide (NAD), both of which are autophagy-promoting events. Accordingly, we demonstrate that scavenging ROS by N-acetylcysteine and repleting NAD independently reduce DNA damage-induced autophagy. In addition, olaparib augmented the effect of DNA-damaging treatment in a human xenograft model of ALL in NOD-scidIL2Rgammanull mice. On the basis of the current findings, we suggest that PARP1 inhibitors may enhance the efficiency of conventional genotoxic therapies and thereby provide a novel treatment strategy for pediatric patients with ALL. IMPLICATIONS: PARP1 inhibitors augment the DNA damage-induced killing of ALL cells by limiting the opposing effects of cAMP-mediated autophagy, which involves ROS-induced PARP1 activation and depletion of cellular NAD levels.

PKA Cß: a forgotten catalytic subunit of cAMP-dependent protein kinase opens new windows for PKA signaling and disease pathologies.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cß proteins are called Cß1, Cß2, Cß3, Cß4 and so-called abc variants of Cß3 and Cß4. Whereas Cß1 is ubiquitously expressed, Cß2 is enriched in immune cells and the Cß3, Cß4 and their abc variants are solely expressed in neuronal cells. All Cα and Cß splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cß splice variants with the exception of Cα1 and Cß1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cß splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies.

Cellular metabolism dictates T cell effector function in health and disease.

In a healthy person, metabolically quiescent T lymphocytes (T cells) circulate between lymph nodes and peripheral tissues in search of antigens. Upon infection, some T cells will encounter cognate antigens followed by proliferation and clonal expansion in a context-dependent manner, to become effector T cells. These events are accompanied by changes in cellular metabolism, known as metabolic reprogramming. The magnitude and variation of metabolic reprogramming are, in addition to antigens, dependent on factors such as nutrients and oxygen to ensure host survival during various diseases. Herein, we describe how metabolic programmes define T cell subset identity and effector functions. In addition, we will discuss how metabolic programs can be modulated and affect T cell activity in health and disease using cancer and autoimmunity as examples.

Consumption of protein-enriched milk has minor effects on inflammation in older adults-A 12-week double-blind randomized controlled trial.

INTRODUCTION: Aging is associated with increased levels of circulating inflammatory markers and reduced muscle mass and strength. OBJECTIVE: We investigated whether intake of protein-enriched milk for 12 weeks would influence markers of inflammation among adults ≥70years of age with reduced physical strength. METHODS: In a double-blind randomized controlled intervention study, subjects were randomly allocated into two groups, receiving a protein-enriched milk (2×20g protein/d, n=14, mean (±SD) age 76.9±4.9 yrs) or an isocaloric carbohydrate drink (n=17, age 77.7±4.8 yrs) for 12 weeks. We measured serum and mRNA expression levels of inflammatory markers in PBMCs. RESULTS: Significant differences in the mRNA expression of nuclear receptor subfamily, group H, member 3 (NR1H3, encoding the LXRα transcription factor) and interferon gamma (INFG) were observed between groups. The mRNA level of TNFRSF1A was significantly reduced, while the mRNA level of dipeptidyl-peptidase 4 (DPP4) was significantly increased, in the control group. The serum level of TNFα increased significantly in the control group, while sTNFRSF1A increased significantly in both groups, but with no significant differences between groups. CONCLUSION: Consumption of a low-fat, protein-enriched milk for 12 weeks had minor effects on inflammatory related markers in older adults compared to an isocaloric carbohydrate drink.

Evolutionary paths of the cAMP-dependent protein kinase (PKA) catalytic subunits.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cß, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cß arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cß kinase family is exceptionally high, a small set of signature residues defining Cα and Cß subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cß clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods.

The testis-specific Cα2 subunit of PKA is kinetically indistinguishable from the common Cα1 subunit of PKA.

BACKGROUND: The two variants of the α-form of the catalytic (C) subunit of protein kinase A (PKA), designated Cα1 and Cα2, are encoded by the PRKACA gene. Whereas Cα1 is ubiquitous, Cα2 expression is restricted to the sperm cell. Cα1 and Cα2 are encoded with different N-terminal domains. In Cα1 but not Cα2 the N-terminal end introduces three sites for posttranslational modifications which include myristylation at Gly1, Asp-specific deamidation at Asn2 and autophosphorylation at Ser10. Previous reports have implicated specific biological features correlating with these modifications on Cα1. Since Cα2 is not modified in the same way as Cα1 we tested if they have distinct biochemical activities that may be reflected in different biological properties. RESULTS: We show that Cα2 interacts with the two major forms of the regulatory subunit (R) of PKA, RI and RII, to form cAMP-sensitive PKAI and PKAII holoenzymes both in vitro and in vivo as is also the case with Cα1. Moreover, using Surface Plasmon Resonance (SPR), we show that the interaction patterns of the physiological inhibitors RI, RII and PKI were comparable for Cα2 and Cα1. This is also the case for their potency to inhibit catalytic activities of Cα2 and Cα1. CONCLUSION: We conclude that the regulatory complexes formed with either Cα1 or Cα2, respectively, are indistinguishable.

Protein kinase A type I activates a CRE-element more efficiently than protein kinase A type II regardless of C subunit isoform.

BACKGROUND: Protein kinase A type I (PKAI) and PKAII are expressed in most of the eukaryotic cells examined. PKA is a major receptor for cAMP and specificity is achieved partly through tissue-dependent expression and subcellular localization of subunits with different biochemical properties. In addition posttranslational modifications help fine tune PKA activity, distribution and interaction in the cell. In spite of this the functional significance of two forms of PKA in one cell has not been fully determined. Here we have tested the ability of PKAI and PKAII formed by expression of the regulatory (R) subunits RIα or RIIα in conjunction with Cα1 or Cß2 to activate a co-transfected luciferace reporter gene, controlled by the cyclic AMP responsive element-binding protein (CREB) in vivo. RESULTS: We show that PKAI when expressed at equal levels as PKAII was significantly (p < 0.01) more efficient in inducing Cre-luciferace activity at saturating concentrations of cAMP. This result was obtained regardless of catalytic subunit identity. CONCLUSION: We suggest that differential effects of PKAI and PKAII in inducing Cre-luciferace activity depend on R and not C subunit identity.

Protein kinase a-dependent phosphorylation of serine 119 in the proto-oncogenic serine/arginine-rich splicing factor 1 modulates its activity as a splicing enhancer protein.

Serine/arginine-rich splicing factor 1 (SRSF1), previously designated SF2/ASF, belongs to a family of SR proteins that regulate constitutive and alternative splicing. SRSF1 expression is increased in tumors from several tissues and elicits changes in key target genes involved in tumor genesis. Several protein kinases phosphorylate SRSF1, which regulates its localization and function. It is previously reported that protein kinase A (PKA) phosphorylates SRSF1, but the importance of this modification is not well characterized. Here, we show that PKA phosphorylates SRSF1 on serine 119 in vitro. Phosphorylation of SRSF1 on this site enhanced the RNA binding capacity of SRSF1 in vivo and reduced the protein's capacity to activate splicing of the Minx transcript in vitro. We also confirm an interaction between SRSF1 and PKA Cα1 and demonstrate that this interaction is not dependent on serine 119 phosphorylation but requires active PKA Cα1. We conclude that PKA phosphorylation of SRSF1 at serine 119 regulates SFRS1-dependent RNA binding and processing but not its interaction with PKA.

Splicing factor arginine/serine-rich 17A (SFRS17A) is an A-kinase anchoring protein that targets protein kinase A to splicing factor compartments.

Protein kinase A (PKA) is targeted to distinct subcellular localizations by specific protein kinase A anchoring proteins (AKAPs). AKAPs are divided into subclasses based on their ability to bind type I or type II PKA or both. Dual-specificity AKAPs were recently reported to have an additional PKA binding determinant called the RI specifier region. A bioinformatic search with the consensus RI specifier region identified a novel AKAP, the splicing factor arginine/serine-rich 17A (SFRS17A). Here, we show by a variety of protein interaction assays that SFRS17A binds both type I and type II PKA in vitro and inside cells, demonstrating that SFRS17A is a dual-specific AKAP. Moreover, immunofluorescence experiments show that SFRS17A colocalizes with the catalytic subunit of PKA as well as the splicing factor SC35 in splicing factor compartments. Using the E1A minigene splicing assay, we found that expression of wild type SFRS17A conferred regulation of E1A alternative splicing, whereas the mutant SFRS17A, which is unable to bind PKA, did not. Our data suggest that SFRS17A is an AKAP involved in regulation of pre-mRNA splicing possibly by docking a pool of PKA in splicing factor compartments.

Isoform-specific regulation of immune cell reactivity by the catalytic subunit of protein kinase A (PKA).

There are two major genes encoding the catalytic subunits of protein kinase A, Calpha and Cbeta. The functional significance of these isoforms is enigmatic. Lymphoid cells of the immune system express both Calpha and Cbeta. In this study we tested the role of Calpha and Cbeta in regulating immune cell reactivity to antigens using mice carrying a targeted disruption of the Calpha and Cbeta gene respectively. Calpha and Cbeta ablation both resulted in a 50% reduction in PKA-specific kinase activity and the level of PKA type I but not PKA type II. Moreover, despite that C subunit ablation did not affect immune cell development and homeostasis, Calpha but not Cbeta ablation augmented expression of the activation marker CD69 on lymphocytes. CD69 induction coincided with immune cell hyperresponsiveness and was associated with reduced sensitivity to cAMP-mediated inhibition of anti-CD3 induced T cell proliferation. Our results imply that Calpha is required for normal immune cell reactivity and demonstrates isoform-specific effects and non-redundant functions of C subunit isoforms expressed in the same cell.

Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit.

BACKGROUND: Epidermal Growth Factor Receptor (EGFR) is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA) induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits. RESULTS: We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Calpha and Cbeta in two different models. The first model used targeted disruption of either Calpha or Cbeta in mice whereas the second model used Calpha and Cbeta RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level. CONCLUSION: Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

Inactive forms of the catalytic subunit of protein kinase A are expressed in the brain of higher primates.

It is well documented that the beta-gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N-terminal end encoded by alternative use of several exons located 5' to exon 2 in the human, bovine and mouse Cbeta gene. In the present study, we demonstrate the expression of six novel human Cbeta mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbetaDelta4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbetaDelta4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbetaDelta4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP-responsive element binding protein. Furthermore, co-expression of CbetaDelta4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CbetaDelta4 variants associate with both RI and RII in a cAMP-independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CbetaDelta4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.

Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cbeta2.

BACKGROUND: Two main genes encoding the catalytic subunits Calpha and Cbeta of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cbeta genes encode several splice variants, including the splice variant Cbeta2. In mouse Cbeta2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cbeta2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. RESULTS: We identified a novel 47 kDa splice variant encoded by the mouse Cbeta gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cbeta2. Based on the present results and the existence of a human brain-specifically expressed Cbeta splice variant designated Cbeta4 that is identical to the former mouse Cbeta2 splice variant, the mouse splice variant has now been renamed mouse Cbeta4. CONCLUSION: Murine lymphoid tissues express a protein that is a homologue of human and bovine Cbeta2. The murine Cbeta gene encodes the splice variants Cbeta1, Cbeta2, Cbeta3 and Cbeta4, as is the case with the human Cbeta gene.

Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression.

We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, approximately 2500 genes are upregulated >3-fold, of which approximately 900 are also expressed in Jurkat cells. Concomitantly, approximately 1500 genes are downregulated or repressed, of which approximately 500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages ( approximately 80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells.

Activation of the cAMP-dependent protein kinase signaling pathway by luteinizing hormone in trout theca layers.

In the fish ovary, LH is the main factor regulating the production of steroids during the periovulatory period and its effects are believed to be mediated, at least partially, through the cAMP-dependent protein kinase (PKA) signaling pathway. However, there is no direct evidence for the presence of PKA in the fish ovary nor on the regulation of its activity by fish LH. Here, we show the identification of regulatory (R) and catalytic (C) subunits of PKA in trout theca cells by immunoblotting. DEAE-cellulose chromatography of theca cell extracts indicated the presence of PKA type I and II and showed that trout theca cells display PKA-specific phosphotransferase and cAMP-binding activities. Salmon LH (sLH) stimulated PKA activity and increased the levels of immunoreactive RIIalpha, RIIbeta and C subunits in trout theca layers. These observations, coupled with the sLH-dependent decrease in the half-life of the C subunit, as shown by pulse-chase experiments, strongly suggest that sLH activates PKA in trout theca cells. Furthermore, our results suggest that ovarian PKA activity and its regulation by LH has been well conserved from fish to humans.

Mutation of the Calpha subunit of PKA leads to growth retardation and sperm dysfunction.

The intracellular second messenger cAMP affects cell physiology by directly interacting with effector molecules that include cyclic nucleotide-gated ion channels, cAMP-regulated G protein exchange factors, and cAMP-dependent protein kinases (PKA). Two catalytic subunits, Calpha and Cbeta, are expressed in the mouse and mediate the effects of PKA. We generated a null mutation in the major catalytic subunit of PKA, Calpha, and observed early postnatal lethality in the majority of Calpha knockout mice. Surprisingly, a small percentage of Calpha knockout mice, although runted, survived to adulthood. This growth retardation was not due to decreased GH production but did correlate with a reduction in IGF-I mRNA in the liver and diminished production of the major urinary proteins in kidney. The survival of Calpha knockout mice after birth is dependent on the genetic background as well as environmental factors, but sufficient adult animals were obtained to characterize the mutants. In these animals, compensatory increases in Cbeta levels occurred in brain whereas many tissues, including skeletal muscle, heart, and sperm, contained less than 10% of the normal PKA activity. Analysis of sperm in Calpha knockout males revealed that spermatogenesis progressed normally but that mature sperm had defective forward motility.