autolysosome; autophagosome; chaperone-mediated autophagy; flux; LC3; lysosome; macroautophagy; phagophore; stress; vacuole
Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.
Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21 expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response–driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency–induced liver NUPR1/FGF21 axis.
Pancreatic ductal adenocarcinoma (PDAC) offers an optimal model for discovering “druggable” molecular pathways that participate in inflammation-associated cancer development. Chronic pancreatitis, a common prolonged inflammatory disease, behaves as a well-known premalignant condition that contributes to PDAC development. Although the mechanisms underlying the pancreatitis-to-cancer transition remain to be fully elucidated, emerging evidence supports the hypothesis that the actions of proinflammatory mediators on cells harboring Kras mutations promote neoplastic transformation. Recent elegant studies demonstrated that the IL-17 pathway mediates this phenomenon and can be targeted with antibodies, but the downstream mechanisms by which IL-17 functions during this transition are currently unclear. In this study, we demonstrate that IL-17 induces the expression of REG3β, a well-known mediator of pancreatitis, during acinar-to-ductal metaplasia and in early PanIN lesions. Furthermore, we found that REG3β promotes cell growth and decreases sensitivity to cell death through activation of the gp130-JAK2-STAT3-dependent pathway. Genetic inactivation of REG3β in the context of oncogenic Kras-driven PDAC resulted in reduced PanIN formation, an effect that could be rescued by administration of exogenous REG3β. Taken together, our findings provide mechanistic insight into the pathways underlying inflammation-associated pancreatic cancer, revealing a dual and contextual pathophysiological role for REG3β during pancreatitis and PDAC initiation.
Patient stratification has been instrumental for the success of targeted therapies in breast cancer. However, the molecular basis of metastatic breast cancer and its therapeutic vulnerabilities remain poorly understood. Here we show that PML is a novel target in aggressive breast cancer. The acquisition of aggressiveness and metastatic features in breast tumours is accompanied by the elevated PML expression and enhanced sensitivity to its inhibition. Interestingly, we find that STAT3 is responsible, at least in part, for the transcriptional upregulation of PML in breast cancer. Moreover, PML targeting hampers breast cancer initiation and metastatic seeding. Mechanistically, this biological activity relies on the regulation of the stem cell gene SOX9 through interaction of PML with its promoter region. Altogether, we identify a novel pathway sustaining breast cancer aggressiveness that can be therapeutically exploited in combination with PML-based stratification.
Targeting PML in acute promyelocytic leukaemia has changed the outcome of patients with this disease. Here, the authors demonstrated that PML is also a potential therapeutic target in breast cancer where it specifically regulates cancer initiating cells and tumour progression through the transcriptional regulation of SOX9.
Pancreatitis is an inflammatory disease that both facilitates and accelerates the transformation of pancreatic cells upon activation of the KRAS oncogene. Autophagy is proposed to be one of the cellular mechanisms contributing to pancreatic carcinogenesis, especially during initial stages in which the KRAS oncogene appears to play a key role. Autophagy is also strongly induced during pancreatitis by the overexpression of VMP1. We recently developed a genetically engineered mouse model in which the VMP1 protein is induced simultaneously with the activation of the oncogene KrasG12D specifically in the pancreas, by the addition of doxycycline to a water drink. Using this sophisticated animal model, we can affirm that pancreatic autophagy, induced during pancreatitis by the overexpression of VMP1, promotes the development of precancerous lesions when induced by the mutated KRAS. In addition, the treatment of these mice with chloroquine, an inhibitor of autophagic flux, reverses the effects of VMP1 in pancreatic cancer induced by the KRAS oncogene. Overall, these results bear both mechanistic and biomedical relevance for further understanding and potentially targeting pathways that are critical for initiating pancreatic carcinogenesis, particularly if associated with pancreatitis.
VMP1; pancreatic cancer; pancreatitis; autophagy; chloroquine; KRAS
During the initiation stage of pancreatic adenocarcinoma induced by oncogenic Kras, pancreatic cells are exposed to both a protumoral effect and an opposing tumor suppressive process known as oncogene-induced senescence. Pancreatitis disrupts this balance in favor of the transforming effect of oncogenes by lowering the tumor suppressive threshold of oncogene-induced senescence through expression of the stress protein Nupr1.
pancreatic cancer; Kras; PanIN; senescence; Nupr1
In a recent article, we found that Tribbles pseudokinase 3 (TRIB3) plays a tumor suppressor role and that this effect relies on the dysregulation of the phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) by the mammalian target of rapamycin complex 2 (mTORC2 complex), and the subsequent hyperphosphorylation and inactivation of the transcription factor Forkhead box O3 (FOXO3).
Tribbles pseudokinases; animal models of cancer; skin carcinogenesis; PTEN; prostate cancer
Activating mutations in the KRAS oncogene are prevalent in pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that pancreatic intraepithelial neoplasia (PanIN) formation, which precedes malignant transformation, associates with the expression of immediate early response 3 (Ier3) as part of a prooncogenic transcriptional pathway. Here, we evaluated the role of IER3 in PanIN formation and PDAC development. In human pancreatic cancer cells, IER3 expression efficiently sustained ERK1/2 phosphorylation by inhibiting phosphatase PP2A activity. Moreover, IER3 enhanced KrasG12D-dependent oncogenesis in the pancreas, as both PanIN and PDAC development were delayed in IER3-deficient KrasG12D mice. IER3 expression was discrete in healthy acinar cells, becoming highly prominent in peritumoral acini, and particularly high in acinar ductal metaplasia (ADM) and PanIN lesions, where IER3 colocalized with phosphorylated ERK1/2. However, IER3 was absent in undifferentiated PDAC, which suggests that the IER3-dependent pathway is an early event in pancreatic tumorigenesis. IER3 expression was induced by both mild and severe pancreatitis, which promoted PanIN formation and progression to PDAC in KrasG12D mice. In IER3-deficient mice, pancreatitis abolished KrasG12D-induced proliferation, which suggests that pancreatitis enhances the oncogenic effect of KRAS through induction of IER3 expression. Together, our data indicate that IER3 supports KRASG12D-associated oncogenesis in the pancreas by sustaining ERK1/2 phosphorylation via phosphatase PP2A inhibition.
Nupr1 is a chromatin protein, which cooperates with KrasG12D to induce PanIN formation and pancreatic cancer development in mice, though the molecular mechanisms underlying this effect remain to be fully characterized. In the current study, we report that Nupr1 acts as a gene modifier of the effect of KrasG12D-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation. Congruently, 5-aza-2′-deoxycytydine, a general inhibitor of DNA methylation, reverses the KrasG12D-induced PanIN development by promoting senescence. This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1–/– mice the expression of KrasG12D induces senescence instead of transformation. Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as KrasG12D, during carcinogenesis. The biomedical relevance of these findings lies in that they support the rational for developing similar therapeutic interventions in human aimed at controlling either the initiation or progression of cancer.
The incidence of pancreatic adenocarcinoma is increasing with more than 43,000 predicted new cases in the US and 65,000 in Europe this year. Pancreatic cancer patients have a short life expectancy with less than 3–4% 5-y survival, which results in an equivalent incidence and mortality rate. One of the major challenges in pancreatic cancer is the identification of pharmacological approaches that overcome the resistance of this cancer to therapy. Intensive research in the past decades has led to the classification of pancreatic cancers and the identification of the driver key genetic events. Despite the advances in understanding the molecular mechanisms responsible for pancreatic cancer pathogenesis, this knowledge had little impact on significantly improving the treatment for this dismal disease. In particular, we know today that the lack of therapeutic response in pancreatic cancer is due to the intrinsic high resistance of these tumors to chemotherapy and radiation, rather than to the inappropriate design of these therapeutic approaches. Thus, in order to ensure a better outcome for pancreatic cancer patients, there is a strong need for research focused on the mechanism that determines this resistant phenotype and the means that might drive enhanced response to therapy.
NUPR1; pancreas cancer; hypoxia; glucose starvation; cannabinoids; AURKA
The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox-driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1-deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS-identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as a molecular regulator of redox-driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research.
autophagy; diabetes; mitochondria; obesity; oxidative stress
Pancreatic adenocarcinoma (PDAC) is a devastating disease with an extremely poor life expectancy and no effective treatment. Autophagy is a process of degradation of cytoplasmic component capable of recycling cellular components or eliminate specific targets. The presence of autophagy in PDAC has been demonstrated. However, the implicated cellular pathways are not fully understood and, more importantly, the role of autophagy in PDAC is matter of intensive debate. This review summarizes recently published data in an attempt to clarify the importance of autophagy in this disease and try to reconcile apparently contradictory results.
Prostate cancer is among the most frequent cancers in men, and despite its high rate of cure, the high number of cases results in an elevated mortality worldwide. Importantly, prostate cancer incidence is dramatically increasing in western societies in the past decades, suggesting that this type of tumor is exquisitely sensitive to lifestyle changes. Prostate cancer frequently exhibits alterations in the PTEN gene (inactivating mutations or gene deletions) or at the protein level (reduced protein expression or altered sub-cellular compartmentalization). The relevance of PTEN in this type of cancer is further supported by the fact that the sole deletion of PTEN in the murine prostate epithelium recapitulates many of the features of the human disease. In order to study the molecular alterations in prostate cancer, we need to overcome the methodological challenges that this tissue imposes. In this review we present protocols and methods, using PTEN as proof of concept, to study different molecular characteristics of prostate cancer.
PTEN; Prostate cancer; Fresh tissue; Molecular biology
Background and aims
PAP/HIP was first reported as an additional pancreatic secretory protein expressed during the acute phase of pancreatitis. It was shown in vitro to be anti‐apoptotic and anti‐inflammatory. This study aims to look at whether PAP/HIP plays the same role in vivo.
A model of caerulein‐induced pancreatitis was used to compare the outcome of pancreatitis in PAP/HIP−/− and wild‐type mice.
PAP/HIP−/− mice showed the normal phenotype at birth and normal postnatal development. Caerulein‐induced pancreatic necrosis was, however, less severe in PAP/HIP−/− mice than in wild‐type mice, as judged by lower amylasemia and lipasemia levels and smaller areas of necrosis. On the contrary, pancreas from PAP/HIP−/− mice was more sensitive to apoptosis, in agreement with the anti‐apoptotic effect of PAP/HIP in vitro. Surprisingly, pancreatic inflammation was more extensive in PAP/HIP−/− mice, as judged from histological parameters, increased myeloperoxidase activity and increased pro‐inflammatory cytokine expression. This result, in apparent contradiction with the limited necrosis observed in these mice, is, however, in agreement with the anti‐inflammatory function previously reported in vitro for PAP/HIP. This is supported by the observation that activation of the STAT3/SOCS3 pathway was strongly decreased in the pancreas of PAP/HIP−/− mice and by the reversion of the apoptotic and inflammatory phenotypes upon administration of recombinant PAP/HIP to PAP/HIP−/− mice.
The anti‐apoptotic and anti‐inflammatory functions described in vitro for PAP/HIP have physiological relevance in the pancreas in vivo during caerulein‐induced pancreatitis.
Arteries and veins modulate cardiovascular homeostasis and contribute to hypertension pathogenesis. Functional differences between arteries and veins are based upon differences in gene expression. To better characterize these expression patterns, and to identify candidate genes that could be manipulated selectively in the venous system, we performed whole genome expression profiling of arteries and veins.
We used the CodeLink platform and the major artery (thoracic aorta) and vein (caudal vena cava) of the rat.
The most prominent difference was pancreatitis associated protein (PAP1), expressed 64-fold higher in vena cava vs aorta. Expression of mRNA for thrombospondins (TSP-1, TSP-4) was greater than 5-fold higher in veins vs arteries. Higher mRNA expression of thrombospondins (TSP-1, 2, 4) and PAP1 in vena cava vs aorta was confirmed by PCR. Immunohistochemical analysis of tissue sections qualitatively confirmed a higher expression of these proteins in vena cava vs aorta.
This is the first gene array study of adult rat arterial and venous tissues, and also the first study to report differences in inflammatory genes between arteries and veins. Data from these studies may provide novel insights into the genetic basis for functional differences between arteries and veins in health and disease.
veins; arteries; gene expression; inflammation
Pancreatitis-associated protein (PAP) was discovered in the pancreatic juice of rats with acute pancreatitis. PAP is a 16 kDa secretory protein structurally related to the C-type lectins although classical lectin-related function has not been reported yet. Then, it was demonstrated that PAP expression may be activated in some tissues in a constitutive or injury- and inflammation-induced manner. More recently, it has been found that PAP acts as an anti-inflammatory factor in vitro and in vivo. PAP expression can be induced by several pro- and anti-inflammatory cytokines and by itself through a JAK/STAT3-dependent pathway. PAP is able to activate the expression of the anti-inflammatory factor SOCS3 through the JAK/STAT3-dependent pathway. The JAK/STAT3/SOCS3 pathway seems to be a common point between PAP and several cytokines. Therefore, it is reasonable to propose that PAP is a new anti-inflammatory cytokine.
Pancreatitis-associated protein; Pancreatitis; Janus kinases; STAT3; SOCS3; Anti-inflammatory; Lectin
AIM: To determine the effects of prophylactic peroxi-some proliferator-activated receptor (PPARγ) agonist administration in an experimental model of post-endoscopic retrograde cholangiopancreatography (post-ERCP) acute pancreatitis.
METHODS: Post-ERCP pancreatitis was induced in male Wistar rats by infusion of contrast medium into the pancreatic duct. In additional group, rosiglitazone, a PPARγ agonist, was administered 1 h before infusion of contrast medium. Plasma and pancreas samples were obtained 6 h after the infusion.
RESULTS: Infusion of contrast medium into the pan-creatic duct resulted in an inflammatory process characterized by increased lipase levels in plasma, and edema and myeloperoxidase activity (MPO) in pancreas. This result correlated with the activation of nuclear factor κB (NFκB) and the inducible NO synthase (iNOS) expression in pancreatic cells. Rosiglitazone reduced the increase in lipase and the level of edema and the increase in myeloperoxidase as well as the activation of NFκB and iNOS expression.
CONCLUSION: A single oral dose of rosiglitazone, given 1 h before post-ERCP pancreatitis induction is effective in reducing the severity of the subsequent inflammatory process. The protective effect of rosiglitazone was associated with NFκB inhibition and the blockage of leukocyte infiltration in pancreas.
Peroxisome proliferator-activated receptor γ; Pancreatitis; Endoscopic retrograde cholangio pancreatography; Inflammation; Nuclear factor κB
We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress-induced autophagy-associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly overexpressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi-based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1-binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon.
During the last couple decades, we have significantly advanced our understanding of mechanisms underlying the development of pancreatic ductural adenocarcinoma (PDAC). In the late 1990s into the early 2000s, a model of PDAC development and progression was developed as a multi-step process associated with the accumulation of somatic mutations. The correlation and association of these particular genetic aberrations with the establishment and progression of PDAC has revolutionized our understanding of this process. However, this model leaves out other molecular events involved in PDAC pathogenesis that contribute to its development and maintenance, specifically those being epigenetic events. Thus, a new model considering the new scientific paradigms of epigenetics will provide a more comprehensive and useful framework for understanding the pathophysiological mechanisms underlying this disease. Epigenetics is defined as the type of inheritance not based on a particular DNA sequence but rather traits that are passed to the next generation via DNA and histone modifications as well as microRNA-dependent mechanisms. Key tumor suppressors that are well established to play a role in PDAC may be altered through hypermethylation, and oncogenes can be upregulated secondary to permissive histone modifications. Factors involved in tumor invasiveness can be aberrantly expressed through dysregulated microRNAs. A noteworthy characteristic of epigenetic-based inheritance is its reversibility, which is in contrast to the stable nature of DNA sequence-based alterations. Given this nature of epigenetic alterations, it becomes imperative that our understanding of epigenetic-based events promoting and maintain PDAC continues to grow.
ArgBP2 (Arg-Binding Protein 2/SORBS2) is an adaptor protein involved in cytoskeleton associated signal transduction, thereby regulating cell migration and adhesion. These features are associated with its antitumoral role in pancreatic cancer cells. Tyrosine phosphorylation of ArgBP2, mediated by c-Abl kinase and counterbalanced by PTP-PEST phosphatase, regulates many of its interactions. However, the exact mechanisms of action and of regulation of ArgBP2 remain largely unknown. We found that ArgBP2 has the capacity to form oligomers which are destabilized by tyrosine phosphorylation. We could show that ArgBP2 oligomerization involves the binding of one of its SH3 domains to a specific proline rich cluster. ArgBP2 self-association increases its binding to some of its molecular partners and decreased its affinity for others. Hence, the phosphorylation/oligomerization state of ArgBP2 directly regulates its functions by modulating its adaptive capabilities. Importantly, using a human pancreatic cancer cell model (MiaPaCa-2 cells), we could validate that this property of ArgBP2 is critical for its cytoskeleton associated functions. In conclusions, we describe a new mechanism of regulation of ArgBP2 where tyrosine phosphorylation of the protein interfere with a SH3 mediated self-interaction, thereby controlling its panel of interacting partners and related functions.
The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 µM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 µM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 µM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered (“fuzzy”), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners.
Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) plays an important role during cell stress response in synergy with the potent “genome-keeper” p53. In human, the gene encoding TP53INP1 is expressed at very high level in some pathological situations, such as inflammation and prostate cancer (PC). TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse, making TP53INP1 a relevant specific target for molecular therapy of Castration Resistant (CR) PC. In that context, detection of TP53INP1 in patient biological fluids is a promising diagnostic avenue. We report here successful development of a new Enzyme-Linked Immunosorbent Assay (ELISA) detecting TP53INP1, taking advantage of molecular tools (monoclonal antibodies (mAbs) and recombinant proteins) generated in the laboratory during the course of basic functional investigations devoted to TP53INP1. The ELISA principle is based on a sandwich immunoenzymatic system, TP53INP1 protein being trapped by a first specific mAb coated on microplate then recognized by a second specific mAb. This new assay allows specific detection of TP53INP1 in serum of several PC patients. This breakthrough paves the way towards investigation of a large cohort of patients and assessment of clinical applications of TP53INP1 dosage.
•TP53INP1 is a key stress-response protein over-expressed in prostate cancer.•We have developed a new ELISA detecting TP53INP1 in serum of prostate cancer patients.•This ELISA will be a powerful tool in investigating value of TP53INP1 as a new biomarker in diagnostic and therapy.
ELISA; p53; TP53INP1; Prostate cancer; Biomarker; aa, amino acid; CR PC, castration resistant prostate cancer; ELISA, enzyme-linked immunosorbent assay; HRP, horse radish peroxydase; SFM, serum free medium; TP53INP1, tumor protein 53-induced nuclear protein 1
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
To assess the possibility of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) as a biological marker for detecting Bladder cancer (BCa), we examined the expression of HIP/PAP in both BCa specimens and BCa cell lines and measured HIP/PAP levels in urine from patients with BCa.
HIP/PAP expression in BCa samples was evaluated by western blot analysis, and urinary levels of HIP/PAP in patients with BCa were measured by enzyme-linked immunosorbent assay. Urine samples were collected from 10 healthy volunteers and 109 with benign urological disorders as controls, and from 101 patients who were diagnosed with BCa.
HIP/PAP was highly expressed in BCa samples as compared with control bladder. Urinary HIP/PAP concentrations were significantly higher in BCa patients than in controls (median value; 3.184 pg/mL vs. 55.200 pg/mL, P <0.0001, by Mann–Whitney U test). Urinary HIP/PAP levels in BCa patients correlated positively with pathological T stages and progression-risk groups among non-muscle invasive BCa (P = 0.0008, by Kruskal-Wallis test). Regarding the recurrence-risk classifications of non-muscle invasive BCa, the urinary levels of HIP/PAP were significantly higher in the intermediate than in the low risk group (P = 0.0002, by Mann–Whitney U test). Based on a cut-off of 8.5 pg/mL, the ability of urinary HIP/PAP levels to detect BCa had a sensitivity of 80.2%, specificity of 78.2%, positive predictive value (PPV) of 75.7%, and negative predictive value (NPV) of 82.3%.
HIP/PAP was abundantly expressed in BCa, and the urinary levels of HIP/PAP could be a novel and potent biomarker for detection of BCa, and also for predicting the risks of recurrence- and progression-risk of non-muscle invasive BCa. A large scale study will be needed to establish the usefulness of this biomarker.
Bladder cancer; Urinary marker; HIP/PAP; ELISA; ROC