Hepsin regulates TGFß signaling via fibronectin proteolysis.

Transforming growth factor-beta (TGFß) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFß activity by acting as a storage compartment of latent-TGFß, but how TGFß is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFß signaling through the release of latent-TGFß from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFß signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFß1, while overexpression of hepsin in mammary tumors increased TGFß signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFß and, importantly, that the ability of hepsin to activate TGFß signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFß pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

Myc-induced AMPK-phospho p53 pathway activates Bak to sensitize mitochondrial apoptosis.

Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellular metabolism to meet the biosynthetic and bioenergetic needs of proliferation. Myc also sensitizes cells to mitochondria-dependent apoptosis. Although metabolic reprogramming has been circumstantially connected to vulnerability to apoptosis, the connecting molecular pathways have remained poorly defined. Here, we show that Myc-induced altered glutamine metabolism involves ATP depletion and activation of the energy sensor AMP-activated protein kinase (AMPK), which induces stabilizing phosphorylation of p53 at Ser15. Under influence of Myc, AMPK-stabilized tumor suppressor protein p53 accumulates in the mitochondria and interacts with the protein complex comprised of B-cell lymphoma 2 (Bcl-2) antagonist/killer (BAK) and Bcl2-like 1 (Bcl-xL). Mitochondrial p53 induces conformational activation of proapoptotic Bak without disrupting the Bak-Bcl-xL interaction. Further liberation of Bak specifically from the p53-activated Bak-Bcl-xL complex leads to spontaneous oligomerization of Bak and apoptosis. Thus, Myc-induced metabolic changes are coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak, demonstrating a cell-intrinsic mechanism to counteract uncontrolled proliferation.

Tumor suppressor function of Liver kinase B1 (Lkb1) is linked to regulation of epithelial integrity.

Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly understood whether genetic alterations corrupting this integrity causally facilitate tumorigenesis. We show that conditional deletion of tumor suppressor gene Lkb1 (Par-4) in the mammary gland compromises epithelial integrity manifested by mislocalization of cell polarity markers, lateralization of tight junctions, deterioration of desmosomes and basement membrane (BM), and hyperbranching of the mammary ductal tree. We identify the desmosomal BM remodelling serine protease Hepsin as a key factor mediating Lkb1 loss-induced structural alterations in mammary epithelium and BM fragmentation. Although loss of Lkb1 alone does not promote mammary tumorigenesis, combination of Lkb1 deficiency with oncogenic c-Myc leads to dramatic acceleration in tumor formation. The results coupling Lkb1 loss-mediated epithelial integrity defects to mislocalization of serine protease Hepsin and to oncogenic synergy with c-Myc imply that Lkb1 loss facilitates oncogenic proliferation by releasing epithelial cells from structural BM boundaries.

Hepsin promotes breast tumor growth signaling via the TGFß-EGFR axis.

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and, downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as the hepatocyte growth factor (HGF) and transforming growth factor beta (TGFß) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of the gene encoding hepsin (Hpn) in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFß and EGFR signaling together with a reduction in epidermal growth factor receptor (EGFR) protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that both basal TGFß signaling and EGFR protein expression are inhibited by neutralizing antibodies or small-molecule inhibitors of hepsin. The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFß and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFß and EGFR pathways in cancer.

Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders.

Oncogenic Ras Disrupts Epithelial Integrity by Activating the Transmembrane Serine Protease Hepsin.

Ras proteins play a causal role in human cancer by activating multiple pathways that promote cancer growth and invasion. However, little is known about how Ras induces the first diagnostic features of invasion in solid tumors, including loss of epithelial integrity and breaching of the basement membrane (BM). In this study, we found that oncogenic Ras strongly promotes the activation of hepsin, a member of the hepsin/TMPRSS type II transmembrane serine protease family. Mechanistically, the Ras-dependent hepsin activation was mediated via Raf-MEK-ERK signaling, which controlled hepsin protein stability through the heat shock transcription factor-1 stress pathway. In Ras-transformed three-dimensional mammary epithelial culture, ablation of hepsin restored desmosomal cell-cell junctions, hemidesmosomes, and BM integrity and epithelial cohesion. In tumor xenografts harboring mutant KRas, silencing of hepsin increased local invasion concomitantly with accumulation of collagen IV. These findings suggest that hepsin is a critical protease for Ras-dependent tumorigenesis, executing cell-cell and cell-matrix pathologies important for early tumor dissemination. SIGNIFICANCE: These findings identify the cell-surface serine protease hepsin as a potential therapeutic target for its role in oncogenic Ras-mediated deregulation of epithelial cell-cell and cell-matrix interactions and cohesion of epithelial structure.

Aberrant differentiation of glutamatergic cells in neocortex of mouse model for fragile X syndrome.

The lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. Our previous studies revealed alterations in the differentiation of FMRP-deficient neural progenitors. Here, we show abnormalities in neurogenesis in the mouse and human embryonic FMRP-deficient brain as well as after in utero transfection of I304N mutated FMRP, which acts in a dominant negative manner in the wild-type mouse brain. Progenitors accumulated abnormally in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) mouse neocortex. An increased density of cells expressing sequentially an intermediate progenitor marker, T-box transcription factor (Tbr2), and a postmitotic neuron marker, T-brain 1 (Tbr1), indicated that the differentiation to glutamatergic cell lineages was particularly disturbed. These abnormalities were associated with an increased density of pyramidal cells of the layer V in the early postnatal neocortex suggesting a role for FMRP in the regulation of the differentiation of neocortical glutamatergic neurons.

Analyzing the Type II Transmembrane Serine Protease Hepsin-Dependent Basement Membrane Remodeling in 3D Cell Culture.

Breakdown of the basement membrane is a key step that precedes tumor invasion, and accumulating evidence suggests a key role for the type II transmembrane proteases (TTSPs) in that process. Overexpression of a TTSP hepsin characterizes many solid cancers, including prostate, breast, and ovarian cancer, and in experimental tumor models, the elevated proteolytic activity of hepsin simultaneously activates several growth factors and cleaves basement membrane protein laminin-332, which is an essential component of the cell-basement membrane junction hemidesmosome. These hepsin-dependent molecular events associate with dramatic loss of basement membrane integrity in mouse tumor models and in three-dimensional (3D) epithelial culture. In particular, the 3D culture systems offer unprecedented possibilities to clarify the mechanistic basis of destructive interactions between out-of-control serine protease activity and the basement membrane structure. Here, we describe how to establish 3D mammary epithelial culture in an exogenous basement membrane-free egg white matrix and provide a protocol for quantitative analysis of the impact of hepsin on laminin-332 and its hemidesmosomal receptor α6-integrin by means of confocal microscopy imaging. These protocols were established to facilitate studies aiming to decipher the exact role of oncogenic proteases in tumor invasion processes and to identify novel therapeutic agents able to intervene these cancer critical processes.

Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform.

Hepsin is a membrane-anchored serine protease whose role in hepatocyte growth factor (HGF) signaling and epithelial integrity makes it a target of therapeutic interest in carcinogenesis and metastasis. Using an integrated design, synthesis, and screening platform, we were able to rapidly develop potent and selective inhibitors of hepsin. In progressing from the initial hit 7 to compound 53, the IC50 value against hepsin was improved from ∼1 µM to 22 nM, and the selectivity over urokinase-type plasminogen activator (uPA) was increased from 30-fold to >6000-fold. Subsequent in vitro ADMET profiling and cellular studies confirmed that the leading compounds are useful tools for interrogating the role of hepsin in breast tumorigenesis.

Protective effect of nisoldipine on dipyridamole-induced myocardial ischemia: correlation with exercise electrocardiography.

BACKGROUND: Nisoldipine, a dihydropyridine calcium channel blocker with strong coronary dilatative action, is commonly used in the treatment of myocardial ischaemia; its beneficial effect on effort angina has been demonstrated by several previous reports. Infusion of dipyridamole in doses sufficient to provoke myocardial ischaemia in patients with significant coronary artery disease is used safely in imaging studies for diagnostic purposes. OBJECTIVE: To evaluate the potential effect of nisoldipine on dipyridamole-induced ischaemia and to compare the results with the effect of nisoldipine on exercise-induced ischaemia. METHOD: Twelve patients (10 men and two women, mean age 62 +/- 8 years) with significant coronary artery disease (at least 70% lumen reduction in at least one major coronary vessel) were selected for inclusion in the study. In accordance with the inclusion criteria, the patients exhibited an ischaemic diagnostic response to a multistage exercise electrocardiography stress test (> 0.15 mV ST segment depression compared with the resting electrocardiographic tracing) and to a dipyridamole-echocardiography test (transient left ventricular dyssynergy of contraction during infusion of dipyridamole up to 0.84 mg/kg over 10 min), after 3 days' cessation of antianginal treatment. After treatment with oral nisoldipine (10 mg twice daily) was introduced, the patients repeated the two tests, within 18 days of the first evaluation. RESULTS: The dipyridamole-echocardiography test was positive for ischaemia in 12 patients who were not receiving nisoldipine and in eight patients who were receiving the drug (100% and 67% respectively, P < 0.05). In the eight patients who gave positive dipyridamole-echocardiography tests both with and without treatment, dipyridamole time (time to onset of dyssynergy during the test) increased from 7.9 +/- 2.9 min to 10.2 +/- 3.1 min (P < 0.01). In these patients, no significant changes were observed, at ischaemia, in the severity and extent of induced dyssynergy, evaluated as wall motion score index (each of 16 left ventricular segments scored from 1 = normal to 4 = dyskinetic) after treatment (score variations from baseline to ischaemia: 0.20 +/- 0.11 without nisoldipine and 0.16 +/- 0.06 with nisoldipine; NS). Variations in dipyridamole time (arbitrarily considered to be 15 min in the negative dipyridamole-echocardiography test) were significantly correlated with variations in exercise time (duration of exercise to exhaustion or diagnostic positive response on the electrocardiogram): r = 0.75 (P < 0.01). No significant differences were recorded in rate-pressure product (beats/min x mmHg x 100) at peak ischaemia between patients who were or were not receiving nisoldipine, during either the exercise electrocardiography stress test (233 +/- 36 with nisoldipine and 244 +/- 39 without nisoldipine; NS) or the dipyridamole-echocardiography test (147 +/- 21 with nisoldipine and 133 +/- 30 without nisoldipine; NS). CONCLUSION: Nisoldipine treatment can protect from dipyridamole-induced ischaemia, being associated with a longer stress time, and completely preventing the development of ischaemia in some patients. The therapy-induced changes in ischaemic threshold during the dipyridamole-echocardiography test correlate with variations in exercise tolerance.

[Propafenone and flecainide in the therapy of ventricular arrhythmias]. / Il propafenone e la flecainide nella terapia delle aritmie ventricolari.

Flecainide and propafenone are antiarrhythmic drugs of the class 1C (Vaughan and Williams) commonly used for ventricular arrhythmias. The purpose of the present study was to evaluate the efficacy of these drugs in 170 consecutive patients with ventricular arrhythmias who referred to our cardiological ambulatory. The study population was divided into two groups according to the absence (group A,82 patients) or presence of organic heart disease (group 1B: 51 patients with left ventricular ejection fraction (LVEF) >35%; group 2B: 37 patients with LVEF<35%). Ventricular arrhythmias were evaluated with a 48 hours Holter monitoring at baseline, and with a control 24 hours Holter monitoring at 15 days (for optimizing the dosage), at 5 months and at 10 months from the beginning of antiarrhythmic therapy. Patients of group A were randomly assigned to antiarrhythmic treatment (flecainide 150-300 mg/die or propafenone 450-900 mg/die). For patients of group B, such choice was leaded by the clinical and strumental data (32 patients were treated with flecainide, 56 patients with propafenone). In the 160 patients who ended the 10 months follow-up, we observed the following results: patients of group A showed a mean percentage reduction in incidence of premature ventricular complexes (PVC) after therapy in comparison to basal conditions of 93% and 89% with flecainide and propafenone, respectively, after a treatment of 5 months (p < 0.001); after 10 months mean percentage reduction of PVC was 91% with each drug (p = n.s.); complex ventricular events (CVE) were reduced of 90% and of 100% after 5 and 10 months, respectively, of treatment with flecainide and of 100% both after 5 and 10 months of treatment with propafenone (p = n.s.) -- patients of group 1B showed a mean percentage reduction of PVC of 87% and 84% after 5 and 10 months, respectiively, of treatment with propafenone (p = n.s.); after 5 months of therapy mean percentage CVE reduction was 66% with flecainide and 86% with propafenone (p < 0.001); after 10 months this mean reduction was 53% with flecainide and 73% with propafenone (p < 0.001). -- patients of group 2B showed a mean reduction of PVC of 59% and 58% after 5 and 10 months of therapy with flecainide, and of 65% and 67% after 5 and 10 months of therapy with propafenone (p = n.s.); CVE were reduced of 28% with flecainide and of 47% with propafenone after 5 months of treatment (p < 0.001) and of 36% with flecainide against 52% with propafenone after 10 months (p < 0.01). In the present study there was no significant difference between the two drugs in terms of tollerance and collateral effects (8% with flecainide vs 7%, with propafenone). Our results confirm the efficacy of the 1C class drugs in the treatment of "essential" ventricular arrhytmias. This efficacy appears reduced in non selected patients with organic heart disease. In these latter patients propafenone has shown more efficacy than flecainide in reducing CVE.

Hyperkinetic ventricular arrhythmias in very elderly people with and without cardiac disease. Correlation with left ventricular echocardiographic parameters.

BACKGROUND: Morphological and functional changes induced by aging can hamper a clear distinction between pathological or paraphysiological phenomena in very old people. The incidence of hyperkinetic ventricular arrhythmias, for example, progressively increases in the elderly, even in the absence of overt cardiac disease. METHODS: One-hundred fifty-two clinically stable patients older than 80 years, submitted within 15 days to clinical evaluation, 24-hour continuous ambulatory ECG monitoring and echo Doppler examination, in the absence of antiarrhythmic treatment, were retrospectively selected in order to evaluate the incidence of ventricular arrhythmias, in patients with and without significant heart disease. The further aim of the study was to correlate the number of arrhythmias with left ventricular morphological and functional parameters, echocardiographically assessed. From the initial population, 80 patients (41 males, age 83 +/- 3 years) had significant heart disease (ischemic, hypertensive or valvular): Group I. Seventy-two patients (30 males, age 83 +/- 3 years) had no clinical or instrumental signs of heart disease: Group II. RESULTS: Considering echocardiographic data, Group I patients had a significantly higher left ventricular end-diastolic diameter (52 +/- 6 mm vs 47 +/- 4 mm, p < 0.01), lower ejection fraction (57 +/- 10% vs 64 +/- 6%, p < 0.01) and higher mass (275 +/- 84 g vs 208 +/- 46 g, p < 0.01), when compared with Group II. From ECG monitoring data, significant differences between the two groups were recorded in the incidence of premature ventricular beats per hour (79 +/- 163 vs 15 +/- 34, Group I vs Group II, p < 0.01) and presence of complex phenomena (couplets, triplets and runs: 51% vs 22%, p < 0.01). In old patients with documented cardiac disease a significant correlation was present between premature ventricular beats incidence and left ventricular end diastolic diameter (r = 0.39, p < 0.05) and left ventricular ejection fraction (r = 0.40, p < 0.05), while in patients without heart disease, no significant correlation was found between incidence of premature ventricular beats and echocardiographic morpho-functional parameters. CONCLUSIONS: In conclusion, hyperkinetic ventricular arrhythmias are globally frequent in old persons of very advanced age (more than 80 years), but, also in this subset, a significant distinction in terms of incidence and severity of arrhythmias is present between subjects with and without cardiac disease. A significant correlation between incidence of premature beats and non-invasive morpho-functional left ventricular parameters is present only for patients with overt heart disease.

Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4.

The PAR clan of polarity regulating genes was initially discovered in a genetic screen searching for genes involved in asymmetric cell divisions in the Caenorhabditis elegans embryo. Today, investigations in worms, flies and mammals have established PAR proteins as conserved and fundamental regulators of animal cell polarization in a broad range of biological phenomena requiring cellular asymmetries. The human homologue of invertebrate PAR-4, a serine-threonine kinase LKB1/STK11, has caught attention as a gene behind Peutz-Jeghers polyposis syndrome and as a bona fide tumour suppressor gene commonly mutated in sporadic cancer. LKB1 functions as a master regulator of AMP-activated protein kinase (AMPK) and 12 other kinases referred to as the AMPK-related kinases, including four human homologues of PAR-1. The role of LKB1 as part of the energy sensing LKB1-AMPK module has been intensively studied, whereas the polarity function of LKB1, in the context of homoeostasis or cancer, has gained less attention. Here, we focus on the PAR-4 identity of LKB1, discussing the weight of evidence indicating a role for LKB1 in regulation of cell polarity and epithelial integrity across species and highlight recent investigations providing new insight into the old question: does the PAR-4 identity of LKB1 matter in cancer?

Faulty epithelial polarity genes and cancer.

Epithelial architecture is formed in tissues and organs when groups of epithelial cells are organized into polarized structures. The epithelial function and integrity as well as signaling across the epithelial layer is orchestrated by apical junctional complexes (AJCs), which are landmarks for PAR/CRUMBS and lateral SCRIB polarity modules and by dynamic interactions of the cells with underlying basement membrane (BM). These highly organized epithelial architectures are demolished in cancer. In all advanced epithelial cancers, malignant cells have lost polarity and connections to the basement membrane and they have become proliferative, motile, and invasive. Clearly, loss of epithelial integrity associates with tumor progression but does it contribute to tumor development? Evidence from studies in Drosophila and recently also in vertebrate models have suggested that even the oncogene-driven enforced cell proliferation can be conditional, dependant on the influence of cell-cell or cell-microenvironment contacts. Therefore, loss of epithelial integrity may not only be an obligate consequence of unscheduled proliferation of malignant cells but instead, malignant epithelial cells may need to acquire capacity to break free from the constraints of integrity to freely and autonomously proliferate. We discuss how epithelial polarity complexes form and regulate epithelial integrity, highlighting the roles of enzymes Rho GTPases, aPKCs, PI3K, and type II transmembrane serine proteases (TTSPs). We also discuss relevance of these pathways to cancer in light of genetic alterations found in human cancers and review molecular pathways and potential pharmacological strategies to revert or selectively eradicate disorganized tumor epithelium.

Overexpression of a truncated TrkB isoform increases the proliferation of neural progenitors.

The truncated isoform of TrkB, TrkB.T1, has been shown to be expressed in the neurogenic region of rodent brain. TrkB.T1 lacks tyrosine kinase activity and it may modify the action of the full-length TrkB. We show here that the full-length TrkB and TrkB.T1 are expressed at the same relative expression levels in mouse neural progenitor cell cultures. The number of neurosphere-forming progenitors was reduced and apoptosis increased in neurospheres generated from mice overexpressing TrkB.T1 when compared with wild-type neurospheres. The proliferation of the transgenic neural progenitors was increased, as indicated by the larger average diameter of spheres (140% of control), the increased cell growth in an MTT assay (137% of control) and the faster rate of 3H-thymidine incorporation (128% of control) in the transgenic cell cultures than in controls. The proliferation of neural progenitors was also increased after lentivirus-mediated TrkB.T1 overexpression. A significant increase in 3H-thymidine incorporation (119% of control) and the average diameter of spheres (112% of control) in the TrkB.T1-transduced neurospheres compared with neurospheres transduced with the control vectors confirmed the role of TrkB.T1 in proliferation of neural progenitor. When induced to differentiate, progenitors overexpressing TrkB.T1 generated two to three times more neurons than did wild-type cells. The increase in the number of neurons correlated with an increase in the number of apoptotic cells (two-fold) at these time points. The data indicate that changes in the relative expression levels of different TrkB isoforms influence the replicative capacity of neural progenitors.

[Left atrial myxoma evidenced by silent acute myocardial infarction]. / Myxoma atriale sinistro manifestatosi con infarto del miocardio acuto silente.

A 64-year-old female patient was in-hospital admitted due to a traumatic femoral fracture. A routinely performed ECG showed signs of anterior acute myocardial infarction, clinically silent, and pathological levels of myocardial serum enzyme were recorded. The echocardiographic-Doppler examination confirmed the LV dyssynergy of contraction and, unexpectedly, revealed a large peduncolated and mobile mass in left atrium, connected to the interatrial septum and prolapsing in left ventricle, referable to myxoma. In the clinical history of the patient, a previous cerebral transitory ischemic attack was present (probably due to myxomatous embolization), but no any other cardiovascular symptoms. The patient successfully underwent coronary angiography, which showed no coronary artery disease, and cardiac surgery for tumoral removal. On the basis of clinical and instrumental data, also acute myocardial infarction may be considered a very likely consequence of a intracoronary embolus. Systemic embolization from left atrial myxomas are frequent; however, the involvement of coronary tree, with clinical manifestations and diagnosis during life, is extremely rare. Complete lack of symptoms due to atrial myxoma and myocardial infarction, and the fortuitous diagnosis of both diseases are peculiar findings of the reported case. Many systemic embolizations from myxomas, although sources of tissue damages, may likely occur without symptoms and may be unrecognized during acute period.