Noncanonical activation of Notch1 protein by membrane type 1 matrix metalloproteinase (MT1-MMP) controls melanoma cell proliferation.

Notch1 is an evolutionarily conserved signaling molecule required for stem cell maintenance that is inappropriately reactivated in several cancers. We have previously shown that melanomas reactivate Notch1 and require its function for growth and survival. However, no Notch1-activating mutations have been observed in melanoma, suggesting the involvement of other activating mechanisms. Notch1 activation requires two cleavage steps: first by a protease and then by γ-secretase, which releases the active intracellular domain (Notch1(NIC)). Interestingly, although ADAM10 and -17 are generally accepted as the proteases responsible of Notch1 cleavage, here we show that MT1-MMP, a membrane-tethered matrix metalloproteinase involved in the pathogenesis of a number of tumors, is a novel protease required for the cleavage of Notch1 in melanoma cells. We find that active Notch1 and MT1-MMP expression correlate significantly in over 70% of melanoma tumors and 80% of melanoma cell lines, whereas such correlation does not exist between Notch1(NIC) and ADAM10 or -17. Modulation of MT1-MMP expression in melanoma cells affects Notch1 cleavage, whereas MT1-MMP expression in ADAM10/17 double knock-out fibroblasts restores the processing of Notch1, indicating that MT1-MMP is sufficient to promote Notch1 activation independently of the canonical proteases. Importantly, we find that MT1-MMP interacts with Notch1 at the cell membrane, supporting a potential direct cleavage mechanism of MT1-MMP on Notch1, and that MT1-MMP-dependent activation of Notch1 sustains melanoma cell growth. Together, the data highlight a novel mechanism of activation of Notch1 in melanoma cells and identify Notch1 as a new MT1-MMP substrate that plays important biological roles in melanoma.

Normal cellular prion protein with a methionine at position 129 has a more exposed helix 1 and is more prone to aggregate.

The human prion gene, PRNP, has two allelic forms that encode either a methionine or valine at codon 129. This polymorphism strongly influences the pathogenesis of prion disease. However, the underlying mechanism remains unclear. We compared the conformation between wild-type human prion protein (rPrP(C)) with either a valine or methionine at position 129, using a panel of monoclonal antibodies that are specific for epitopes along the entire protein. We found that rPrP(C(129M)) has a more exposed helix 1 region compared to rPrP(C(129V)). Helix 1 is important in the aggregation process. Accordingly, rPrP(C(129M)) aggregates at a faster rate and forms more aggregate than rPrP(C(129V)). In addition, by using a rPrP with a pathogenic mutation of five additional octapeptide repeat insertions, rPrP((129M)/10OR), as "seeds", we showed that rPrP((129M)/10OR) promotes the aggregation of rPrP(C(129M)) more efficiently than rPrP(C(129V)). These findings provide a possible mechanism underlying the influence of residue 129 on human prion disease.

Normal cellular prion protein is a ligand of selectins: binding requires Le(X) but is inhibited by sLe(X).

The normal PrP(C) (cellular prion protein) contains sLe(X) [sialyl-Le(X) (Lewis X)] and Le(X). sLe(X) is a ligand of selectins. To examine whether PrP(C) is a ligand of selectins, we generated three human PrP(C)-Ig fusion proteins: one with Le(X), one with sLe(X), and the other with neither Le(X) nor sLe(X). Only Le(X)-PrP(C)-Ig binds E-, L- and P-selectins. Binding is Ca(2+)-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLe(X)-PrP(C)-Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrP(C). The selectins precipitated PrP(C) in human brain in a Ca(2+)-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrP(C) precipitated. Therefore the presence of sialic acid prevents the binding of PrP(C) in human brain to selectins. Hence, human brain PrP(C) interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences.

Aggregation of prion protein with insertion mutations is proportional to the number of inserts.

Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.

Synchronized Targeting of Notch and ERBB Signaling Suppresses Melanoma Tumor Growth through Inhibition of Notch1 and ERBB3.

Despite significant advances in melanoma therapy, melanoma remains the deadliest form of skin cancer, with a 5-year survival rate of only 15%. Thus, novel treatments are required to address this disease. Notch and ERBB are evolutionarily conserved signaling cascades required for the maintenance of melanocyte precursors. We show that active Notch1 (Notch1(NIC)) and active (phosphorylated) ERBB3 and ERBB2 correlate significantly and are similarly expressed in both mutated and wild-type BRAF melanomas, suggesting these receptors are co-reactivated in melanoma to promote survival. Whereas blocking either pathway triggers modest effects, combining a ?-secretase inhibitor to block Notch activation and a tyrosine kinase inhibitor to inhibit ERBB3/2 elicits synergistic effects, reducing cell viability by 90% and hampering melanoma tumor growth. Specific inhibition of Notch1 and ERBB3 mimics these results, suggesting these are the critical factors triggering melanoma tumor expansion. Notch and ERBB inhibition blunts AKT and NF?B signaling. Constitutive expression of NF?B partially rescues cell death. Blockade of both Notch and ERBB signaling inhibits the slow cycling JARID1B-positive cell population, which is critical for long-term maintenance of melanoma growth. We propose that blocking these pathways is an effective approach to treatment of melanoma patients regardless of whether they carry mutated or wild-type BRAF.

MT1-MMP modulates melanoma cell dissemination and metastasis through activation of MMP2 and RAC1.

Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1-MMP is a membrane-associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion-promoting, pro-tumorigenic MMP in cancer. We show that deregulation of MT1-MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1-MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1-MMP in melanoma pathogenesis. We demonstrate that MT1-MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1-MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1-MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1-MMP is required to sustain RAC1 activity and promote MT1-MMP-dependent cell motility. These data highlight a novel MT1-MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.

An ERBB3/ERBB2 oncogenic unit plays a key role in NRG1 signaling and melanoma cell growth and survival.

We recently identified neuregulin-1 (NRG1) as a novel target of Notch1 required in Notch-dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1-dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1-ERBB3-ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.

Ligand binding promotes prion protein aggregation--role of the octapeptide repeats.

Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu(2+) and Zn(2+). Here, we report our findings that GAG and Cu(2+) promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu(2+), mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild-type PrP. When the GAG-binding motif, KKRPK, is deleted the effect of GAG but not that of Cu(2+) is abolished. By contrast, when the Cu(2+)-binding motif, the octapeptide-repeat region, is deleted, neither GAG nor Cu(2+) is able to promote aggregation. Therefore, the octapeptide-repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti-PrP mAbs, whereas none of the tested anti-PrP mAbs block Cu(2+)-promoted aggregation. However, a mAb that is specific for an epitope at the N-terminus enhances aggregation in the presence of either GAG or Cu(2+). Therefore, although binding of either GAG or Cu(2+) promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation.

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans.

Mutation in the prion gene PRNP accounts for 10-15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrP(C)). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrP(C). Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.

Test for detection of disease-associated prion aggregate in the blood of infected but asymptomatic animals.

We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP(Sc). However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

Prion proteins with insertion mutations have altered N-terminal conformation and increased ligand binding activity and are more susceptible to oxidative attack.

We compared the biochemical properties of a wild type recombinant normal human cellular prion protein, rPrP(c), with a recombinant mutant human prion protein that has three additional octapeptide repeats, rPrP(8OR). Monoclonal antibodies that are specific for the N terminus of rPrP(c) react much better with rPrP(8OR) than rPrP(c), suggesting that the N terminus of rPrP(8OR) is more exposed and hence more available for antibody binding. The N terminus of PrP(c) contains a glycosaminoglycan binding motif. Accordingly, rPrP(8OR) also binds more glycosaminoglycan than rPrP(c). In addition, the divalent cation copper modulates the conformations of rPrP(c) and rPrP(8OR) differently. When compared with rPrP(c), rPrP(8OR) is also more susceptible to oxidative damage. Furthermore, the abnormalities associated with rPrP(8OR) are recapitulated, but even more profoundly, in another insertion mutant, which has five extra octapeptide repeats, rPrP(10OR). Therefore, insertion mutants appear to share common features, and the degree of abnormality is proportional to the number of insertions. Any of these anomalies may contribute to the pathogenesis of inherited human prion disease.

Biochemical fingerprints of prion infection: accumulations of aberrant full-length and N-terminally truncated PrP species are common features in mouse prion disease.

Infection with any one of three strains of mouse scrapie prion (PrPSc), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrPSc strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrPSc are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrPSc aggregates, indicating that they are physically associated with PrPSc. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrPSc infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.

An aggregation-specific enzyme-linked immunosorbent assay: detection of conformational differences between recombinant PrP protein dimers and PrP(Sc) aggregates.

The conversion of the normal cellular prion protein, PrP(C), into the protease-resistant, scrapie PrP(Sc) aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP(Sc) aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP(Sc) aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.