Protease-Activated Drug Development

In this extensive review, we elucidate the importance of proteases and their role in drug development in various diseases with an emphasis on cancer. First, key proteases are introduced along with their function in disease progression. Next, we link these proteases as targets for the development of prodrugs and provide clinical examples of protease-activatable prodrugs. Finally, we provide significant design considerations needed for the development of the next generation protease-targeted and protease-activatable prodrugs

Failed immune responses across multiple pathologies share pan-tumor and circulating lymphocytic targets

Tumor-infiltrating lymphocytes (TILs) are widely associated with positive outcomes, yet carry key indicators of a systemic failed immune response against unresolved cancer. Cancer immunotherapies can reverse their tolerance phenotypes while preserving tumor reactivity and neoantigen specificity shared with circulating immune cells. We performed comprehensive transcriptomic analyses to identify gene signatures common to circulating and TILs in the context of clear cell renal cell carcinoma. Modulated genes also associated with disease outcome were validated in other cancer types. Through comprehensive bioinformatics analyses, we identified practical diagnostic markers and actionable targets of the failed immune response. On circulating lymphocytes, 3 genes (LEF1, FASLG, and MMP9) could efficiently stratify patients from healthy control donors. From their associations with resistance to cancer immunotherapies and microbial infections, we uncovered not only pan-cancer, but pan-pathology, failed immune response profiles. A prominent lymphocytic matrix metallopeptidase cell migration pathway is central to a panoply of diseases and tumor immunogenicity, correlates with multi-cancer recurrence, and identifies a feasible noninvasive approach to pan-pathology diagnoses. The differentially expressed genes we have identified warrant future investigation into the development of their potential in noninvasive precision diagnostics and precision pan-disease immunotherapeutics. - 2019, American Society for Clinical Investigation.We thank all study participants and patients; The Cancer Genome Atlas; Mathieu Latour and Roula Albadine and supporting staff of the CHUM pathology department; Manon de Ladurantaye and Anne-Marie Mes-Masson from the CRCHUM for RNA quality profiling, Geneviève Cormier and Fred Saad from the CRCHUM for drawing blood from control donors; Gilles Corbeil of the CRCHUM genomics department for RNA quality testing and microarray profiling; Francois Harvey of the CRCHUM bioinformatics department; Peter Graf and Patrick Sabourin from Affymetrix for providing reagents and technical assistance; Zeeshan Farroq and Ofir Goldberger from Fluidigm; Erika Diaz from StemCell; Andrew Mouland from McGill University; Simon Turcotte from University of Montreal; and Sascha Ring from Biostars for their advice. This work was partially performed at the Institut du Cancer de Montréal CRCHUM and University of Montreal, in Montreal, Quebec, Canada. This work was supported by a Canadian Cancer Society Research Institute grant (CCSRI) (702036, to RL and IJ) and a Biomedical Research Grant from the Kidney Foundation of Canada (KFOC130019 to RL). RL is supported by the Quebec Cell, Tissue and Gene Therapy Network—ThéCell (a thematic network supported by the Fonds de recherche du Québec–Santé [FRQS]), the FRQS, and the Immunotherapy Network (iTNT) from the Terry Fox Research Institute (TFRI), A. Monette is supported by Mitacs, Merck, l’Institut du cancer de Montréal (ICM), the Society for Immunotherapy of Cancer, and the Lady Davis Institute for Medical Research. NAB is supported by the FRQS post-doctoral award and Qatar University. JBL is supported by l’Institut du Cancer de Montréal. JPR holds the Louis Lowenstein Chair, McGill University. DEK is supported by an FRQS Research Scholar Award (grant 31035), CIHR 377124, NHLBI RO1-HL-092565, and the Canada Foundation for Innovation (CFI) (grant 31756). IJ and computational analysis were supported by the Canada Research Chair Program (CRC) (grant 225404), Ontario Research Fund (grant 34876), the Natural Sciences Research Council (NSERC) (grant 203475), the CFI (grants 203373 and 30865), the Krembil Foundation, and IBM.Scopu

Ennusteeseen vaikuttavia kasvainmerkkiaineita paksu- ja peräsuolisyövässä

Background and aims: The most important prognostic factor in colorectal cancer (CRC) is tumour stage. Prognosis of local tumours is good, but in tumours with lymph node or distant metastasis, the prognosis is worse. Patients with stage III (Dukes C) tumours usually receive adjuvant chemotherapy. Patients with stage IV (Dukes D) tumours cannot be treated curatively by surgery alone and usually receive chemotherapy. In stage II (Dukes B) disease, adjuvant chemotherapy is recommended for patients at risk for recurrence, such as with tumours with vascular or perineural invasion, or in cases with emergency surgery or insufficient lymph-node harvest. In order to identify better those patients requires additional prognostic factors like biomarkers. Material and methods: Clinical data came from 643 consecutive patients who underwent surgery for colorectal cancer at the Department of Surgery, Meilahti Hospital, Helsinki University Central Hospital, between 1982 and 1998. Clinical data and archival tissue specimens were available from 623 cases. For MMP-9, a validation series of 213 patients treated between 1998 and 2001 was included. Survival data came from the Population Register Centre of Finland and Statistics Finland. Tissue microarray (TMA) blocks were prepared from re-evaluated histological archive blocks. TMA slides were stained with MMP-2, MMP-7, MMP-8, MMP-9, TATI, trypsinogen-1, trypsinogen-2, p53, Ki-67, and EGFR antibodies. Correlation of immunoexpression of markers with clinicopathological variables was assessed. Survival analysis was performed by the Kaplan-Meier method, and multivariate Cox proportional hazards model. Results: Study I showed strong MMP-7 to be an independent prognostic marker for 5-year survival, but later the difference faded. In Study II, no association was observable between p53 or Ki-67 expression and survival. In Study III, TATI immunoexpression was an independent prognostic marker for improved survival, particularly in subgroups of trypsinogen-1- and trypsinogen-2-positive patients, although trypsinogen-1 and trypsinogen-2 were not prognostic factors. In Study IV, MMP-9 expression was an independent prognostic marker of favourable survival in Dukes B patients, but the validation series did not confirm these results. MMP-2 and MMP-8 immunoexpression lacked any correlation with prognosis. In Study V, EGFR+TATI+ patients had significantly better prognosis than did those with EGFR+TATI-, EGFR-TATI+, or EGFR-TATI-. Conclusion: MMP-7, MMP-9, TATI, and the TATI-EGFR combination can all serve as prognostic biomarkers in CRC.Tausta: Tärkein paksu- ja peräsuolisyövän ennustetta arvioiva tekijä on kasvaimen levinneisyys (stage). Paikallisissa kasvaimissa ennuste on hyvä, mutta imusolmukkeisiin tai laajemmin levinneissä kasvaimissa ennuste on huonompi. Imusolmukkeisiin levinneessä (stage III) paksu- ja peräsuolisyövässä käytetään liitännäissolusalpaajahoitoa. Stage IV kasvaimia, joissa tauti on lähettänyt etäpesäkkeitä muihin elimiin, pelkästään kirurgisella hoidolla ei päästä parantavaan lopputulokseen, vaan näitä potilaita hoidetaan sytostaattilääkityksellä. Stage II levinneisyydessä kasvain työntyy suolen lihaskerrokseen, mutta ei ole lähettänyt etäpesäkkeitä. Tässä ryhmässä liitännäissytostaatteja suositellaan käytettäväksi kasvaimissa, joihin liittyy uusiutumisriskiä lisääviä ominaisuuksia, kuten kasvaimen työntyminen suoni- tai hermorakenteisiin, jos leikkaus on tehty tukostilanteessa, tai jos suoliliepeen poistettujen imusolmukkeiden määrä on riittämätön. Jotta uusiutumisriskissä olevat potilaat havaittaisiin paremmin, tarvitaan lisää ennustetta tarkentavia apuvälineitä, kuten kasvainmerkkiaineita. Menetelmät: Tutkimuksen potilasaineisto koostui 643:stä vuosina 1982-1998 Helsingin yliopistollisen keskussairaalan Meilahden yksikössä operatiivisesti hoidetusta paksu- ja peräsuolisyöpäpotilaasta. Kliiniset tiedot ja kudosnäytteet olivat saatavana 623:sta potilaasta. Lisäksi MMP-9-työssä käytettiin vertailusarjaa, joka koostui 213:sta potilaasta vuosilta 1998-2001. Elossaolotiedot saatiin Väestörekisteristä ja Tilastokeskuksesta. Kasvaimista valmistettiin nk. tissue microarray (TMA)-näytteet, jotka värjättiin immunohistokemiallisella menetelmällä. Tutkitut kasvainmerkkiaineet olivat MMP-2, MMP-7, MMP-8, MMP-9, TATI, trypsinogeeni-1, trypsinogeeni-2, p53, Ki-67 ja EGFR. Immunoekspression ja kliinispatologisten muuttujien välinen korrelaatio analysoitiin. Eloonjäämisanalyysinä käytettiin Kaplan-Meierin menetelmää sekä Cox:n monimuuttuja-analyysia. Tulokset: Osatyö I:ssä todettiin voimakkaan MMP-7-ekspression olevan itsenäinen ennustetekijä viiden vuoden seuranta-aikana, mutta pidemmällä seuranta-aikavälillä eroa ei enää voitu osoittaa. Osatyö II:ssa todettiin, että p53- ja Ki-67-ekspressio ei vaikuta ennusteeseen. Osatyö III:ssa osoitettiin TATI-ekspression olevan itsenäinen ennustetekijä ja liittyvän parantuneeseen ennusteeseen. Erityisesti trypsinogeeni-1-ja trypsinogeeni-2-positiivisilla potilailla TATI oli voimakas ennustetekijä. Trypsinogeeni-1-tai trypsinogeeni-2-ekspressio ei vaikuttanut potilaiden ennusteeseen. Osatyö IV:ssä MMP-9-ekspressio osoittautui itsenäiseksi hyvän ennusteen merkkiaineeksi stage II potilailla, tosin vertailuaineistossa tulosta ei saatu vahvistettua. MMP-2:n ja MMP-8:n immunoekspressio ei vaikuttaneet potilaiden ennusteeseen. Osatyö V:ssä EGFR+TATI+ potilailla oli merkitsevästi parempi ennuste kuin EGFR+TATI-, EGFR-TATI+ tai EGFR-TATI- potilailla. Yhteenveto: MMP-7:a, MMP-9:a, TATI:a ja TATI-EGFR-yhdistelmää voidaan käyttää ennusteen arvioimisessa paksu- ja peräsuolisyöpäpotilailla

Matrix metalloproteinases in colorectal cancer development and prognosis

Colorectal cancer (CRC) is the second leading cause of cancer death in the Netherlands and the fourth worldwide. Matrix metalloproteinases (MMPs) are involved in the process of colorectal cancer development and progression. MMPs are capable of degrading the extracellular matrix components of the intestinal basement membrane and facilitate invasion into the deeper layers of the bowel wall, lymph nodes and/or blood vessels. Furthermore, they are implicated in several processes in the microenvironment of colorectal cancer, like angiogenesis, cell death and inflammation. In this thesis, the focus is on the clinical impact of matrix metalloproteinases (MMPs) in the different stages of colorectal cancer development and metastasis. MMP-7, -8 and -9 were shown to be involved in the early stages of colorectal cancer development, whereas MMP-2 levels were only increased in cancer tissue but not in precancerous lesions. Furthermore, MMP-2, MMP-7 and MMP-9 were identified as predictors of outcome in patients with colorectal carcinoma, both at genetic (SNP) and protein level. The increased knowledge of the role of MMPs in the various stages of CRC might contribute to further development of specific anti-MMP therapies in the future.UBL - phd migration 201

Relationship among expression of basic-fibroblast growth factor, MTDH/Astrocyte elevated gene-1, adenomatous polyposis coli, matrix metalloproteinase 9,and COX-2 markers with prognostic factors in prostate carcinomas

Background: The etiopathogenesis of prostate cancer (PC) is still not clear, but hormonal, genetic, and environmental factors are thought to play a role in the tumor pathogenesis. Astrocyte elevated gene-1(AEG-1) as a novel transmembrane protein is predominantly located in the perinuclear region and endoplasmic reticulum. It has been found that AEG-1 upregulation increases the invasive ability of glioma and prostate cancer. Basic fibroblast growth factor (bFGF), matrix metalloproteinase-9 (MMP-9), cyclooxygenases-2 (COX-2), and adenomatous polyposis coli (APC) are very important in tumor progression as well. Materials and Methods: This study included 97 radical prostatectomy specimens. IHC stains for bFGF, MMP-9, COX-2, APC, and AEG-1 were performed on the tissue microarray using standard procedures. For each patient, the age, Gleason score, tumor volume, lymphovascular invasion, lymph node metastasis, surgical margin, and the invasion of vesiculoseminalis areas were assessed. Analyses were performed using the statistical PASW (ver. 18). Results: Statistically significant positive relationships were found MMP-9 and COX-2 (r = 0.242 and P = 0.017), between MMP-9 and APC (r = 0.207 and P = 0.043), and between bFGF and AEG-1 (r = 0.295 and P = 0.004). However, the relationships between age and staining results and tumor volume and staining results were not found to be significant. Although a positive correlation was found between the Gleason score and tumor volume and the Gleason score and age (r = 0.415 and P = 0.0001; r = 0.246 and P = 0.015, respectively), we did not find a statistically significant relationship between other stains and other prognostic parameters (lymphovascular invasion, lymph node metastasis, surgical margin, or vesiculoseminalis invasion). Conclusion:The relationships we found between MMP-9 and COX-2, between MMP-9, and APC and between bFGF and AEG-1 as independent prognostic parameters could be helpful in the development of new therapeutic procedures.Keywords: Adenomatous polyposis coli, astrocyte elevated gene‑1, basic fibroblast growth factor, cyclooxygenases‑2, matrix metalloproteinase‑9, prognostic parameters, prostate adenocarcinomasNigerian Journal of Clinical Practice • Oct-Dec 2013 • Vol 16 • Issue

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.Matriksin metalloproteinaasit (MMP:t) ovat 23-jäseninen entsyymiperhe, jotka pystyvät pilkkomaan käytännössä kaikkia soluvälitilan proteiineja. Ne jaetaan rakenteen perusteella 8 alaryhmään ja substraattispesifisyyden perusteella 6 ryhmään. MMP:ien toiminta on liitetty tulehdukseen, kudostuhoon, solujen vaeltamiseen, niveltulehdukseen, verisuonten uudelleenrakentumiseen ja uudismuodostukseen, sekä kasvaimien kasvuun ja leviämiseen. MMP:ien toimintaa estävät niiden luonnolliset estäjät (TIMP:t). Eri MMP:ien toiminta riippuu kohdekudoksesta. Väitöskirjatyössäni tutkin viime vuosina eristettyjen MMP:ien toimintaa suoliston tulehduksissa, suolisto- ja ihohaavan paranemisessa sekä ruoansulatuskanavan syöpäsairauksissa. Tutkin useita MMP:eja ja TIMP:eja selvittääkseni niiden sijainnin kudostasolla ja lisätäkseni tietoa MMP:ien mahdollisesta toiminnasta eri solutyypeissä ja sairauksissa: terve suolisto, tulehduksellinen suolistosairaus, keskoslasten nekrotisoiva enterokoliitti, pyoderma gangrenosum-ihosairaus, ja paksusuolensyöpä sekä haimasyöpä. Lisäksi etsin mahdollisia merkkiaineita, joilla keliakiaoireisten lasten keliakia-diagnoosi voitaisiin varmistaa ennen diagnostisia löydöksiä ohutsuolikoepalassa. Päätutkimusmenetelminä käytettiin immunohistokemiaa, in situ-hybridisaatiota ja Taqman RT-PCR:ää. Tulokseni osoittavat, että MMP-26:a (matrilysiini-2) tarvitaan suolisto- ja ihohaavan paranemiseen. Paksusuolisyövässä ja haimasyövässä MMP-26 vaikuttaisi olevan sopiva merkkiaine levinneisyyden arviointiin, vaikka se ei itse ilmennykään leviämisrintamassa. MMP-21 esiintyminen lisääntyy haimasyövässä ja liittyy mahdollisesti kasvaimen erilaistumiseen. MMP-19 ja -28 (epilysiini) liittyvät suoliston limakalvon normaaliin uusiutumiseen, mutta ne häviävät kudoksesta paksusuolisyövässä ikään kuin ne olisivat suojelevia merkkiaineita . MMP-12 (metalloelastaasi) on olennainen proteaasi suoliston tulehduksissa ja kudostuhossa, kuten on huomattu aikaisemmissa keliakiatöissä ja nyt nekrotisoivassa enterokoliitissa. Nuoruusiän diabetesta sairastavilla MMP-1, -3 ja -12 ilmentyivät suoliston limakalvolla enemmän kuin kontrolleilla. MMP-7 on voimakkaasti koholla kudostasolla nekrotisoivassa enterokoliitissa. Huonosti paranevassa haavassa, jollainen pyoderma gangrenosum on, MMP-8, -9 ja -10 kuten TNFα saattavat lisätä haavapedin proteiinien tuhoa. MMP-1 sekä MMP-26 puuttuminen keratinosyyteistä taasen hidastaa haavan epitelisaatiota. Tuloksieni perusteella MMP-26 sopii huonon ennusteen merkkiaineeksi haimasyövässä ja paksusuolisyövässä mutta se toimii eri tavalla eri kudoksissa ja kasvaintyypeissä, joten tätä päätelmää ei voida yleistää. Lisäksi MMP-26 on hyödyllinen merkkiaine hyvin paraneville haavoille, mikäli sitä löytyy haavan reunan soluista. Jotta MMP-12 ilmentyminen voidaan todeta hyväksi merkkiaineeksi myöhemmin ilmentyvälle keliakialle suolioireisilla potilailla, tarvitsemme vielä lisätutkimuksia laajemmalla potilasmäärällä. MMP-7 on yksi olennaisimmista proteaaseista nekrotisoivan enterokoliitin aiheuttamassa kudostuhossa, joten sille spesifien proteaasinestäjien tutkiminen olisi aiheellista. Pyoderma gangrenosumissa TNFα-estäjät ovat sopivia lääkkeitä myös histologiselta kannalta, mikä onkin jo osoitettu kliinisissä kokeissa. Totean että usean MMP:n tutkimista tarvitaan eri sairauksissa ja terveissä kudoksissa, jotta voimme ymmärtää näiden proteaasien toimintaa kudostasolla. MMP:t tai TIMP:t eivät ainoastaan tuhoa tai korjaa kudosrakenteita. Ne näyttävät toimivan eri kudoksissa eri tavalla. Löytääksemme tehokkaita MMP:ien täsmäestäjiä hoitotarkoituksiin, meidän on ymmärrettävä MMP:ien profiili ja niiden toiminta eri elimissä ettemme, häiritsisi normaaleja korjaavia toimintoja haavan paranemisessa tai valkosolujen suorittamassa ensi vaiheen syöpäsolujen torjunnassa