A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CßII.

Serine protease 14 (Prss14)/epithin is a transmembrane serine protease that plays essential roles in tumor progression and metastasis and therefore is a promising target for managing cancer. Prss14/epithin shedding may underlie its activity in cancer and worsen outcomes; accordingly, a detailed understanding of the molecular mechanisms in Prss14/epithin shedding may inform the design of future cancer therapies. On the basis of our previous observation that an activator of PKC, phorbol 12-myristate 13-acetate (PMA), induces Prss14/epithin shedding, here we further investigated the intracellular signaling pathway involved in this process. While using mitogen-activated protein kinase inhibitors to investigate possible effectors of downstream PKC signaling, we unexpectedly found that an inhibitor of c-Jun N-terminal kinase (JNK), SP600125, induces Prss14/epithin shedding even in the absence of PMA. SP600125-induced shedding, like that stimulated by PMA, was mediated by tumor necrosis factor-α-converting enzyme. In contrast, a JNK activator, anisomycin, partially abolished the effects of SP600125 on Prss14/epithin shedding. Moreover, the results from loss-of-function experiments with specific inhibitors, short hairpin RNA-mediated knockdown, and overexpression of dominant-negative PKCßII variants indicated that PKCßII is a major player in JNK inhibition- and PMA-mediated Prss14/epithin shedding. SP600125 increased phosphorylation of PKCßII and tumor necrosis factor-α-converting enzyme and induced their translocation into the plasma membrane. Finally, in vitro cell invasion experiments and bioinformatics analysis of data in The Cancer Genome Atlas breast cancer database revealed that JNK and PKCßII are important for Prss14/epithin-mediated cancer progression. These results provide important information regarding strategies against tumor metastasis.

Intramembrane proteolysis of an extracellular serine protease, epithin/PRSS14, enables its intracellular nuclear function.

BACKGROUND: Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein. RESULTS: In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b. CONCLUSIONS: These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment.

Influences of ethanol and temperature on sucrose-evoked response of gustatory neurons in the hamster solitary nucleus.

Taste-responsive neurons in the nucleus of the solitary tract (NST), the first gustatory nucleus, often respond to thermal or mechanical stimulation. Alcohol, not a typical taste modality, is a rewarding stimulus. In this study, we aimed to investigate the effects of ethanol (EtOH) and/or temperature as stimuli to the tongue on the activity of taste-responsive neurons in hamster NST. In the first set of experiments, we recorded the activity of 113 gustatory NST neurons in urethane-anesthetized hamsters and evaluated responses to four basic taste stimuli, 25% EtOH, and 40°C and 4°C distilled water (dH2O). Sixty cells responded to 25% EtOH, with most of them also being sucrose sensitive. The response to 25% EtOH was significantly correlated with the sucrose-evoked response. A significant correlation was also observed between sucrose- and 40°C dH2O-and between 25% EtOH- and 40°C dH2O-evoked firings. In a subset of the cells, we evaluated neuronal activities in response to a series of EtOH concentrations, alone and in combination with 32 mM sucrose (EtOH/Suc) at room temperature (RT, 22°C-23°C), 40°C, and 4°C. Neuronal responses to EtOH at RT and 40°C increased as the concentrations increased. The firing rates to EtOH/Suc were greater than those to EtOH or sucrose alone. The responses were enhanced when solutions were applied at 40°C but diminished at 4°C. In summary, EtOH activates most sucrose-responsive NST gustatory cells, and the concomitant presence of sucrose or warm temperatures enhance this response. Our findings may contribute to elucidate the neural mechanisms underlying appetitive alcohol consumption.

Shedding of epithin/PRSS14 is induced by TGF-ß and mediated by tumor necrosis factor-α converting enzyme.

Epithin/PRSS14, a type II transmembrane serine protease, plays critical roles in cancer metastasis. Previously, we have reported that epithin/PRSS14 undergoes ectodomain shedding in response to phorbol myristate acetate (PMA) stimulation. In this study, we show that transforming growth factor-ß (TGF-ß) induces rapid epithin/PRSS14 shedding through receptor mediated pathway in 427.1.86 thymoma cells. Tumor necrosis factor-α converting enzyme (TACE) is responsible for this shedding. Amino acid sequence encompassing the putative shedding cleavage site of epithin/PRSS14 exhibit strong homology to the cleavage site of l-selectin, a known TACE substrate. TACE inhibitor, TAPI-0 and TACE siRNA greatly reduced TGF-ß-induced epithin/PRSS14 shedding. TGF-ß treatment induces translocation of intracellular pool of TACE to the membrane where epithin/PRSS14 resides. These findings suggest that TGF-ß induces epithin/PRSS14 shedding by mediating translocation of epithin/PRSS14 sheddase, TACE, to the membrane.

Environment-Sensitive Ectodomain Shedding of Epithin/PRSS14 Increases Metastatic Potential of Breast Cancer Cells by Producing CCL2.

Epithin/PRSS14 is a membrane serine protease that plays a key role in tumor progression. The protease exists on the cell surface until its ectodomain shedding, which releases most of the extracellular domain. Previously, we showed that the remaining portion on the membrane undergoes intramembrane proteolysis, which results in the liberation of the intracellular domain and the intracellular domainmediated gene expression. In this study, we investigated how the intramembrane proteolysis for the nuclear function is initiated. We observed that ectodomain shedding of epithin/PRSS14 in mouse breast cancer 4T1 cells increased depending on environmental conditions and was positively correlated with invasiveness of the cells and their proinvasive cytokine production. We identified selenite as an environmental factor that can induce ectodomain shedding of the protease and increase C-C motif chemokine ligand 2 (CCL2) secretion in an epithin/PRSS14-dependent manner. Additionally, by demonstrating that the expression of the intracellular domain of epithin/PRSS14 is sufficient to induce CCL2 secretion, we established that epithin/PRSS14- dependent shedding and its subsequent intramembrane proteolysis are responsible for the metastatic conversion of 4T1 cells under these conditions. Consequently, we propose that epithin/PRSS14 can act as an environment-sensing receptor that promotes cancer metastasis by liberating the intracellular domain bearing transcriptional activity under conditions promoting ectodomain shedding.

Targeting metastatic breast cancer with peptide epitopes derived from autocatalytic loop of Prss14/ST14 membrane serine protease and with monoclonal antibodies.

BACKGROUND: In order to develop a new immunotherapeutic agent targeting metastatic breast cancers, we chose to utilize autocatalytic feature of the membrane serine protease Prss14/ST14, a specific prognosis marker for ER negative breast cancer as a target molecule. METHODS: The study was conducted using three mouse breast cancer models, 4 T1 and E0771 mouse breast cancer cells into their syngeneic hosts, and an MMTV-PyMT transgenic mouse strain was used. Prss14/ST14 knockdown cells were used to test function in tumor growth and metastasis, peptides derived from the autocatalytic loop for activation were tested as preventive metastasis vaccine, and monoclonal and humanized antibodies to the same epitope were tested as new therapeutic candidates. ELISA, immunoprecipitation, Immunofluorescent staining, and flow cytometry were used to examine antigen binding. The functions of antibodies were tested in vitro for cell migration and in vivo for tumor growth and metastasis. RESULTS: Prss14/ST14 is critically involved in the metastasis of breast cancer and poor survival rather than primary tumor growth in two mouse models. The epitopes derived from the specific autocatalytic loop region of Prss14/ST14, based on structural modeling acted as efficient preventive metastasis vaccines in mice. A new specific monoclonal antibody mAb3F3 generated against the engineered loop structure could reduce cell migration, eliminate metastasis in PyMT mice, and can detect the Prss14/ST14 protein expressed in various human cancer cells. Humanized antibody huAb3F3 maintained the specificity and reduced the migration of human breast cancer cells in vitro. CONCLUSION: Our study demonstrates that Prss14/ST14 is an important target for modulating metastasis. Our newly developed hybridoma mAbs and humanized antibody can be further developed as new promising candidates for the use in diagnosis and in immunotherapy of human metastatic breast cancer.

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats.

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCbeta2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

Glutamate-induced cobalt uptake elicited by kainate receptors in rat taste bud cells.

Glutamate-induced cobalt uptake reveals non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) in rat taste bud cells. However, it is not known which type of non-NMDA glutamate receptors is involved. We used a cobalt staining technique combined with pharmacological tests for kainate or alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors and/or immunohistochemistry against subunits of GluRs to examine the presence of non-NMDA receptors in rat foliate tastebud cells. Cobalt uptake into taste cells was elicited by treating taste buds with glutamate, kainate or SYM 2081, a kainate receptor agonist. Treating taste buds with AMPA or fluorowillardiine did not stimulate significant cobalt uptake. Moreover, 6-cyano-7-nitro-quinoxaline-2, 3-dione significantly reduced cobalt staining elicited by glutamate or kainate receptor agonists, but SYM 2206, an AMPA receptor antagonist, did not. Immunohistochemistry against subunits of GluRs reveals GluR6 and KA1-like immunoreactivity. Moreover, most glutamate-induced cobalt-stained cells showed GluR6 and KA1-like immunoreactivity. These results suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs.