Versican Proteolysis Research Articles

Versican Proteolytic Fragments (Matrikines) Regulate the Intratumoral Dendritic Cell Milieu In Vivo: Implications for in Situ Tumor Vaccination

Regulated proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) through the actions of ADAMTS-proteases, is associated with enhanced CD8+ infiltration in both hematopoietic and solid tumors. However, it is unclear whether the enhanced CD8+ infiltration results from proteolysis-mediated depletion of precursor VCAN at the tumor site or from generation of bioactive proteolytic fragments ("matrikines") (e.g., the 441-aa N-terminal fragment of V1-VCAN isoform, versikine). We have previously shown that versikine promotes Batf3-dendritic cell (DC) generation from FLT3L-mobilized bone marrow (BM) progenitors in vitro. However, the effects of versikine in DC homeostasis in the tumor microenvironment in vivo are unknown. To investigate the effects of versikine in DC homeostasis in vivo, we utilized the first Ras-driven myeloma (MM) model (VQ model- Rajagopalan et al., Blood 132:1006, 2018) as well as transplantable solid tumor models in both C57BL/6J (LLC lung carcinoma) and Balb/c (4T1 mammary carcinoma) backgrounds. Tumor cells were stably engineered to secrete HA-tagged versikine vs. empty-vector (EV) controls. EV-VQ or versikine-VQ myeloma cells were implanted intracardiacally into C57BL/6J syngeneic recipients and mice were monitored until they developed myeloma-related end-organ damage (hindlimb paralysis). Both groups of mice were paralyzed at similar rates. Intratumoral conventional DCs (CD138-CD45+, CD11chi,MHC IIhiLy6C-, CD64-) clustered into two populations: cDC1 (Batf3-DC: CD24hi,CD11blo), a subset with crucial activity in cross-priming anti-tumor CD8+ T cells, and cDC2 (CD24lo, CD11bhi). Versikine enhanced intratumoral Batf3-DC frequency/infiltration, while cDC2 levels were diminished in versikine-VQ BM (Figure 1A)(Batf3-DC: 48% in EV-VQ vs. 72% in versikine-VQ, p-value= 0.0246; cDC2: 52% in EV-VQ vs. 28% in versikine-VQ, p=0.0312). Monocytic-derived DC (Mo-DC: CD11chi, MHC IIhi, Ly6C+, CD64+) frequency remained unchanged. Versikine's effects were replicated in 2 solid tumor models. Versikine-expressing tumors were characterized by significantly enhanced Batf3-DC infiltration (Fig. 1A, p-value= 0.0079 for 4T1 model and <0.0001 for LLC model), whereas cDC2 numbers were diminished (p-value: 0.0079 and <0.0001 respectively). Adoptive transfer of CD45.2+ pre-DC (SIRPaint, FLIT3+, CD11c+, MHC II-, Celltrace+) in LLC-EV and LLC-versikine tumors in CD45.1+ recipients did not show any differences in 3-day differentiation potential of DC precursors, implicating other mechanisms to explain the steady-state imbalance in DC subset frequencies. To examine whether versikine's effects on the intratumoral DC milieu in vivo could be therapeutically harnessed, we compared responses to STING agonist therapy between versikine-expressing and EV tumors. LLC-EV-OVA and LLC-versikine-OVA (ovalbumin, a model antigen) -expressing tumors received therapeutic intratumoral injections of DMXAA, a murine STING agonist. Analysis of splenocytes 5 days later showed a significant increase in the frequency of OVA antigen-specific, CD8+ (MHCI:SIINFEKL tetramer+) splenocytes in LLC-versikine-bearing animals (Figure 1B). Interestingly, there was a marked increase in total central memory T splenocytes (TCM) (CD62LhiCD44hi) harvested from LLC-versikine tumor-bearing mice. We conclude that versikine influences the DC milieu in the tumor bed with promotion of intratumoral cross-presenting Batf3-DC and depletion of the cDC2 subset. Our findings highlight an unappreciated facet of immune regulation of the tumor microenvironment through matrix proteolytic fragments ("matrikines"). Whereas detection of native VCAN proteolysis on myeloma biopsies (see abstract by Dhakal et al.in this meeting) portends adverse outcomes likely due to the tolerogenic effects of accumulated precursor VCAN at the tumor site, therapeutic use of the isolated, purified fragments may promote tumor innate sensing and effector priming. VCAN-matrikines, through their effects on intratumoral Batf3-DC and antigen-specific CD8+ T cell infiltration, may potentiate in situ vaccination strategies across diverse hematopoietic and solid tumor types. Figure 1 Disclosures No relevant conflicts of interest to declare.

Exosites in Hypervariable Loops of ADAMTS Spacer Domains control Substrate Recognition and Proteolysis

ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M−1 s−1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M−1 sec−1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739–744 and 837–844) and ADAMTS-4 (residues 717–724 and 788–795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.

Abstract 1909: Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma

Abstract Background: Clinical responses to checkpoint inhibitors in gastrointestinal cancers have largely been limited to MSI-high disease. Proteolysis of an immunosuppressive proteoglycan versican (VCAN) by ADAMTS proteases correlates with enhanced CD8+ T cell infiltration in colorectal cancer independent of mismatch repair status. Here we investigate the potential of this biomarker in pancreatic ductal adenocarcinoma (PDAC) as VCAN is abundant VCAN in this disease. Methods: KPC mice were used to generate mouse PDAC tissue and organotypic spheroids. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissues across 131 patients. IHC was used to determine levels of VCAN, an immunostimulatory fragment versikine (Vkine), and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were derived from BALB/c mice. VCAN-specific ADAMTS protease expression profiles were compared between antitumor (M1) and protumor (M2) mouse macrophages, human pancreatic stellate cells (PSCs) and patient-derived PDAC-organotypic spheroids. Results: IHC analysis of normal murine pancreas demonstrated low VCAN and Vkine staining. VCAN is abundant in metaplastic/neoplastic KPC mouse pancreatic tissues. Rare VCAN proteolytic predominant areas (samples with both low VCAN and high Vkine) in KPC tumors had increased tumor-infiltrating CD8+ T cells/ high-powered field compared to weak areas (17 vs 4, p<0.001). Human PDACs had abundant VCAN expression compared to normal pancreatic tissues (p<0.001, n=118). Vkine was absent from normal tissues and present only at low levels in tumor cores (p<0.001, n=117). No human PDAC VCAN proteolytic-predominant samples were identified. M1 BMDMs demonstrated high relative expression (RE) of VCAN-specific ADAMTS proteases relative to unpolarized M0 BMDMs; however, these levels decreased under KPC-conditioned media (ADAMTS4, 101 vs 5, p=0.003; ADAMTS9, 80 vs 1, p=0.02; ADAMTS15, 103 versus 1.6, p<0.001; ADAMTS20, 881 vs 3.2, p<0.001). Similarly, M2 BMDMs had elevated ADAMTS-levels that were reduced under KPC-conditioned media, (ADAMTS4, 42 vs 0.4, p=0.0029; ADAMTS9, 40 vs 1, p=0.002; ADAMTS15, 23 vs 3, p=0.027; ADAMTS20, 310 vs 0.8, p<0.001; n=3). RE levels of ADAMTS proteases in KPC spheroids was consistently lower than reported above in macrophages. RE of ADAMTS proteases in human PSCs were between 18 and 304. No differences in ADAMTS expression were identified under PDAC-conditioned media in the human PSCs (p=0.3). Conclusions: The VCAN proteolysis predominant phenotype correlates with increased CD8+ T cell infiltration in PDAC though is rarely present in the human disease. PDAC epithelial cells influence the stromal cells to reduce VCAN proteolysis through decreased ADAMTS expression by macrophages. Future studies examining measures to enhance VCAN proteolysis could result in a more immune permissive microenvironment. Citation Format: Philip B. Emmerich, Chelsie Sievers, Hanna Rainiero, Rosabella Pitera, Connor Maloney, Susan Payne, Mitchell Depke, Cheri Pasch, Linda Clipson, Jillian K. Johnson, Kristina Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1909.

Abstract 4583: Versican accumulation and proteolysis in neuroendocrine tumors

Abstract Introduction: Neuroendocrine tumors (NETs) are a rare, understudied form of human cancer lacking robust preclinical models. Our laboratory has recently demonstrated the importance of the tumor microenvironment (TME) in T-cell exclusion from tumors. Here we examine the accumulation of the immunoregulatory proteoglycan, versican (VCAN), and its immunostimulatory proteolysis product, versikine (Vkine), produced by VCAN cleavage via ADAMTS proteins. Immunotherapeutics are increasingly being studied in NETs and investigation of the immune permissive nature of their TME is warranted. Methods: Human NET tissue microarrays (TMAs) were obtained from the University of Wisconsin Carbone Cancer Center TSB Biobank, containing human NET tissues across a broad spectrum of disease sites including the lung and gastrointestinal (GI) tract among others. Immunohistochemistry was performed using antibodies to VCAN, Vkine, and CD8+ T-cells. VCAN and Vkine levels were measured using a four-tiered relative intensity system (0/1/2/3), while CD8+ T-cell infiltration was measured by counting the CD8+ T-cells per high power field (hpf). Experimental groups were binned as low if VCAN or Vkine scored 0 or 1 and high if VCAN or Vkine scored 2 or 3. Results: Across all samples, high VCAN levels were observed in 20% of samples while high Vkine levels were observed in 55% of samples. Within the lung NET subset, which includes small cell lung cancer, 15% of samples were designated VCAN high and 65% were Vkine high. For the GI NET subset, 34% of samples scored as VCAN high while 50% scored as Vkine high. This study identifies a greater amount of Vkine high tumors present in NETs than we previously observed for colorectal, anal, pancreatic, and breast cancers. The proteolytic predominant phenotype (VCAN low and Vkine high) was present in 55% of lung NETs and 31% of GI NETs. CD8+ T cell infiltration correlated with the VCAN proteolysis predominant phenotype (VCAN low/Vkine High - CD8+ T-cell 16/hpf vs. all other VCAN/Vkine phenotypes 6.5/hpf, p<0.001). This trend persisted across all NET specimen types, including lung and GI primary NETs. Discussion: VCAN accumulation occurs in a subset of NETs, likely leading to immune exclusion and potential resistance to immunotherapies. Excitingly, we demonstrate that a large subset of these cancers has active proteolysis of VCAN in the TME leading to release of the immunostimulatory fragment Vkine. Those NETs with low levels of VCAN and high levels of Vkine are predicted to have a permissive TME for immunotherapies. Further studies are needed to discern the underlying mechanisms resulting in this proteolysis and the signaling consequences with Vkine production. Additionally, this study indicates that VCAN proteolysis should be investigated as a novel biomarker for immunotherapy response for NETs. Citation Format: Christopher P. Babiarz, Philip B. Emmerich, Carley M. Sprackling, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican accumulation and proteolysis in neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4583.

Abstract 3146: Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer

Abstract Background: Infiltration of CD8+ T cells has shown important prognostic implications in colorectal cancers (CRC). Our group has recently correlated proteolysis of an extracellular matrix proteoglycan, versican (VCAN), with infiltration of CRCs by CD8+ T cells. Cleavage by ADAMTS proteases generates a bioactive fragment of versican, versikine (VKINE). VKINE stimulates Batf3-driven dendritic cell activation and maturation, which are critical for immunotherapy efficacy. Methods: CT26 murine CRC cells were transfected to constitutively express VKINE. BALB/c mice between 50 and 70 days of age were orthotopically injected with either 105 empty vector (CT26-EV) or VKINE-expressing (CT26-VKINE) cells. Tumor growth was tracked weekly with colonoscopy and growth rate assessed using percent lumen occlusion. Mice were euthanized and dissected once moribund. Tissues were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemical (IHC) staining was performed using standard procedures. Additionally, FFPE samples from human CRC liver metastases (n=9) were obtained under the University of Wisconsin Translational Science Biocore Institutional Review Board-approved protocol and IHC performed for VCAN, VKINE and CD8. The number of CD8+ tumor-infiltrating lymphocytes (TILs) per high-powered field (HPF) within the malignant epithelium was calculated using four separate 400x magnification fields of view. Results: CT26-EV and CT26-VKINE injected mice both had a ~70% tumor initiation rate and these tumors averaged ~50% lumen occlusion by 14 days post injection. Survival data for each group were compared using a log-rank test. A trend towards improved survival was observed in those mice injected with CT26-VKINE (average survival of 27 days for CT26-VKINE compared to 22 days for CT26-EV, p=0.41, n=13). The average number of CD8+ TILs for each tumor were compared. For mice with CT26-VKINE, the average number of CD8+ TILs per HPF was 7.6, compared to 4.7 for mice with CT26-EV (p=0.39). Human CRC liver metastases were analyzed for VCAN, VKINE, and CD8+ T cells. Several lesions had little to no VCAN staining, whereas others had intense pockets or peripheral staining. VKINE staining was more abundant in metastatic CRC lesions than in primary lesions. CD8+ T cells were excluded in the setting of increased VCAN staining and infiltration was noted specifically in areas of VCAN proteolysis (low VCAN and high VKINE staining). These areas of proteolysis were more often present at the tumor margin and correlated with ADAMTS1 expression. Conclusions: Immunotherapies offer a unique treatment option for cancer patients, but their use is limited in CRC. VCAN proteolysis and the generation of bioactive VKINE has potential to influence the ability of CD8+ T cells to infiltrate the tumor in both primary and metastatic CRC, indicating the potential to utilize VCAN and VKINE as immune biomarkers or therapeutic targets. Citation Format: Philip B. Emmerich, Susan N. Payne, Connor J. Maloney, Rosabella T. Pitera, Hanna Rainiero, Gioia Sha, Athanasios T. Papadas, Adam C. Pagenkopf, Michael F. Bassetti, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3146.

ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation

SUMMARYFocal adhesions anchor cells to extracellular matrix (ECM) and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC) focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM.

Keynote 290: Pembrolizumab (pem) in combination with stereotactic body radiotherapy for resectable liver metastatic colorectal cancer.

TPS874 Background: Colorectal cancer (CRC) is one of the few cancers in which aggressive local approaches to treat systemic metastases have resulted in clinical benefit. Despite these approaches 70-80% of patients (pts) with resectable metastatic disease will recur. The one-year recurrence rate for this population is ~40-50%. Immunotherapeutics have yet to be studied in this setting. To sensitize microsatellite stable (MSS) CRCs to immune therapies adjunctive therapies are required. Gross tumor microenvironments suppress systemic anti-cancer immune responses. Stereotactic body radiation therapy (SBRT) is commonly utilized to treat liver metastatic CRC, using very high doses of focused radiation to ablate tumors. This causes localized inflammation, a potential increase in immunogenic intra-tumoral and intra-lymphatic antigen release, and a rapid influx of responding immune cells. We hypothesize that radiotherapy-induced tumor necrosis, and surgical removal of the remaining immunosuppressive gross disease will enhance the immunogenicity of CRC and potentiate the effectiveness of PD-1 blockade. Methods: This is a single arm phase 1b investigator-initiated study (NCT02837263) to evaluate the use of pem in combination with ablative radiotherapy for the treatment of oligometastatic CRC. This study examines the sequential use of SBRT, then pem followed by resection of all known sites of disease and 6 months of adjuvant pem. Pts with MSS CRC with resectable liver metastatic disease who have received prior FOLFOX are eligible. The primary endpoints are safety/tolerability and the rate of cancer recurrence at 1 year. Secondary endpoints include time to disease recurrence, disease free survival and overall survival. This study is currently enrolling with a planned total accrual of 15 patients. Correlative studies examine PD-L1 status, tumor infiltrating lymphocytes, evidence of an abscopal effect, versican proteolysis as a novel immune biomarker, and the ability of multiparametric fluorodeoxyglucose (FDG) PET MR and fluorothymidine (FLT) PET CT to detect immune infiltration. Clinical trial information: NCT02837263.

Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer.

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8+ T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8+ T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103+CD11chiMHCIIhi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow-derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents.

Versican G1 domain enhances adenoviral-mediated transgene expression and can be modulated by inhibitors of the Janus kinase (JAK)/STAT and Src family kinase pathways

To examine the biochemical influences that may contribute to the success of gene therapy for ocular disorders, the role of versican, a vitreous component, in adenoviral-mediated transgene expression was examined. Versican is a large chondroitin sulfate-containing, hyaluronic acid-binding proteoglycan present in the extracellular matrix and in ocular vitreous body. Y79 retinoblastoma cells and CD44-negative SK-N-DZ neuroblastoma cells transduced with adenoviral vectors in the presence of versican respond with an activation of transgene expression. Proteolysis of versican generates a hyaluronan-binding G1 domain. The addition of recombinant versican G1 to SK-N-DZ cells results in a similar activation of transgene expression, and treatment with dasatinib, an inhibitor of Src family kinases, also mimics the effects of versican. Enhancement is accompanied by an increase in signal transducer and activator of transcription 5 (STAT5) phosphorylation and is abrogated by treatment with C188-9, a STAT3/5 inhibitor, or with ruxolitinib, a Janus kinase 1/2 (JAK1/2) inhibitor. These data implicate versican G1 in enhancing adenoviral vector transgene expression in a hyaluronic acid-CD44 independent manner that is down-regulated by inhibitors of the JAK/STAT pathway and enhanced by inhibitors of the Src kinase pathway.

Versican proteolysis as a key regulator of CD8+ T-cell infiltration in colorectal cancer.

e15082 Background: Colorectal cancer (CRC) originates within immunologically complex microenvironments. To date the benefits of immunotherapy have been modest except in neoantigen-laden mismatch repair (MMR)-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes (TILS) in the tumor bed may substantially augment clinical immunotherapy responses. Proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) generates a bioactive fragment, versikine, with putative immunostimulatory activities. Methods: Matched normal and CRC tissue samples were collected from 122 patients with cancers across all stages and locations throughout the colon and rectum. These samples were stained for VCAN, αDPEAAE (neoepitope generated in cleaving VCAN to versikine), and CD8 and scored by a pathologist. Tumors were classified as VCAN proteolysis-predominant (VPP) if their staining for total VCAN staining intensity was < 1+ and staining for VCAN proteolysis (αDPEAAE antibody) was > 2. Conversely, tumors were classified as VCAN proteolysis-weak (VPW) if intact VCAN staining intensity was > 1+ or αDPEAAE intensity was < 2+. IHC for mismatch repair (MMR) proteins was also performed. Results: Overall increased VCAN staining was observed in cancer versus (vs) normal tissue. VPP tumors had a 10 fold greater infiltration of CD8+ T-cells vs VPW cancers (p < 0.001). The correlation between VCAN proteolysis and CD8+ T-cell infiltration was maintained in both cancers with proficient (p) MMR and deficient (d) MMR. In both pMMR and dMMR, the VPP tumors had the greatest degree of CD8+ T-cell infiltration (Wilcoxon rank sum tests: pMMR p = 0.006; dMMR p = 0.03). Among the VPP tumors there was a greater degree of CD8+ T cell infiltration in the dMMR cancers vs pMMR cancers (35 versus 14.8 TILs per high power filed, p = 0.04). Nuclear CTNNB1, a marker for activation of WNT signaling, negatively correlated with CD8+ T cell infiltration( p = 0.014). In addition, VCAN accumulation correlated with the presence of nuclear CTNNB1 (p < 0.001) Conclusions: This is the first description indicating that VCAN proteolysis may shape CRC immune contexture and provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker.

Versican proteolysis is associated with robust CD8+ T-cell infiltration in human mismatch repair-proficient and -deficient colorectal cancers

Abstract Colorectal cancer (CRC) originates within immunologically complex microenvironments. To date the benefits of immunotherapy have been modest except in neoantigen-laden mismatch repair (MMR)-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. We recently reported that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN), in myeloma tumors, generates a bioactive fragment, versikine, with putative immunostimulatory activities. We show that VCAN proteolysis strongly correlated with CD8+ T-cell infiltration in CRC. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T-cell infiltration. The correlation between VCAN proteolysis and CD8+ T-cell infiltration was maintained in MMR-proficient and -deficient CRCs. Tumor-intrinsic WNT pathway activation was associated with CD8+ T-cell exclusion and correlated with VCAN accumulation. Our findings indicate VCAN proteolysis may shape CRC immune contexture and provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker. Therapeutic manipulation of the VCAN-versikine axis may augment immunotherapy efficacy against CRC.

Versican Proteolysis by ADAMTS Proteases and Its Influence on Sex Steroid Receptor Expression in Uterine Leiomyoma.

Leiomyomas have abundant extracellular matrix (ECM), with upregulation of versican, a large proteoglycan. We investigated ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs) protease-mediated versican cleavage in myometrium and leiomyoma and the effect of versican knockdown in leiomyoma cells. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and RNA in situ hybridization for analysis of myometrium, leiomyoma and immortalized myometrium and leiomyoma cells. Short interfering RNA (siRNA) was used to knockdown versican in leiomyoma cells. This study was performed in an academic laboratory. Study subjects were women with symptomatic or asymptomatic leiomyoma. We quantified messenger RNAs (mRNAs) for versican splice variants. We identified ADAMTS-cleaved versican in myometrium and leiomyoma and ADAMTS messenger RNAs and examined the effect of VCAN siRNA on smooth muscle differentiation and expression of estrogen and progesterone receptors. The women in the symptomatic group (n = 7) had larger leiomyoma (P = 0.01), heavy menstrual bleeding (P < 0.01), and lower hemoglobin levels (P = 0.02) compared with the asymptomatic group (n = 7), but were similar in age and menopausal status. Versican V0 and V1 isoforms were upregulated in the leiomyomas of symptomatic versus asymptomatic women (P = 0.03 and P = 0.04, respectively). Abundant cleaved versican was detected in leiomyoma and myometrium, as well as in myometrial and leiomyoma cell lines. ADAMTS4 (P = 0.03) and ADAMTS15 (P = 0.04) were upregulated in symptomatic leiomyomas. VCAN siRNA did not effect cell proliferation, apoptosis, or smooth muscle markers, but reduced ESR1 and PR-A expression (P = 0.001 and P = 0.002, respectively). Versican in myometrium, leiomyomas and in the corresponding immortalized cells is cleaved by ADAMTS proteases. VCAN siRNA suppresses production of estrogen receptor 1 and progesterone receptor-A. These findings have implications for leiomyoma growth.

Versican proteolysis in the pathogenesis of uterine leiomyoma

Versican proteolysis in the pathogenesis of uterine leiomyoma

Immunoregulatory roles of versican proteolysis in the myeloma microenvironment

AbstractMyeloma immunosurveillance remains incompletely understood. We have demonstrated proteolytic processing of the matrix proteoglycan, versican (VCAN), in myeloma tumors. Whereas intact VCAN exerts tolerogenic activities through Toll-like receptor 2 (TLR2) binding, the immunoregulatory consequences of VCAN proteolysis remain unknown. Here we show that human myeloma tumors displaying CD8+ infiltration/aggregates underwent VCAN proteolysis at a site predicted to generate a glycosaminoglycan-bereft N-terminal fragment, versikine. Myeloma-associated macrophages (MAMs), rather than tumor cells, chiefly produced V1-VCAN, the precursor to versikine, whereas stromal cell–derived ADAMTS1 was the most robustly expressed VCAN-degrading protease. Purified versikine induced early expression of inflammatory cytokines interleukin 1β (IL-1β) and IL-6 by human myeloma marrow-derived MAMs. We show that versikine signals through pathways both dependent and independent of Tpl2 kinase, a key regulator of nuclear factor κB1-mediated MAPK activation in macrophages. Unlike intact VCAN, versikine-induced Il-6 production was partially independent of Tlr2. In a model of macrophage-myeloma cell crosstalk, versikine induced components of “T-cell inflammation,” including IRF8-dependent type I interferon transcriptional signatures and T-cell chemoattractant CCL2. Thus the interplay between stromal cells and myeloid cells in the myeloma microenvironment generates versikine, a novel bioactive damage-associated molecular pattern that may facilitate immune sensing of myeloma tumors and modulate the tolerogenic consequences of intact VCAN accumulation. Therapeutic versikine administration may potentiate T-cell–activating immunotherapies.

ADAMTS9-Mediated Extracellular Matrix Dynamics Regulates Umbilical Cord Vascular Smooth Muscle Differentiation and Rotation.

Despite the significance for fetal nourishment in mammals, mechanisms of umbilical cord vascular growth remain poorly understood. Here, the secreted metalloprotease ADAMTS9 is shown to be necessary for murine umbilical cord vascular development. Restricting it to the cell surface using a gene trap allele, Adamts9(Gt), impaired umbilical vessel elongation and radial growth via reduced versican proteolysis and accumulation of extracellular matrix (ECM). Both Adamts9(Gt) and conditional Adamts9 deletion revealed that ADAMTS9 produced by mesenchymal cells acted non-autonomously to regulate smooth muscle cell (SMC) proliferation, differentiation, and orthogonal reorientation during growth of the umbilical vasculature. In Adamts9(Gt/Gt), we observed interference with PDGFRβ signaling via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which regulates cytoskeletal dynamics during SMC rotation. In addition, we observed disrupted Shh signaling and perturbed orientation of the mesenchymal primary cilium. Thus, ECM dynamics is a major influence on umbilical vascular SMC fate, with ADAMTS9 acting as its principal mediator.

Determinants of Versican-V1 Proteoglycan Processing by the Metalloproteinase ADAMTS5

Proteolysis of the Glu(441)-Ala(442) bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu(441) is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu(441)-Ala(442) site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser(507) and Ser(525) as essential for processing of the Glu(441)-Ala(442) bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser(507) and Ser(525) is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu(441) and an antibody to a peptide spanning Thr(432)-Gly(445) (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu(441)-Ala(442). V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu(441). Therefore, versican cleavage can be inhibited substantially by mutation of Glu(441), Ser(507), and Ser(525) or by an antibody to the region of the scissile bond.

Isolation and purification of versican and analysis of versican proteolysis.

Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. It is crucial for several developmental processes in the embryo and there is increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and a versican fragment provided here, are likely to facilitate further progress on the biology of versican and its proteolysis.

ADAMTS4 and ADAMTS5 knockout mice are protected from versican but not aggrecan or brevican proteolysis during spinal cord injury.

The chondroitin sulfate proteoglycans (CSPGs) aggrecan, versican, and brevican are large aggregating extracellular matrix molecules that inhibit axonal growth of the mature central nervous system (CNS). ADAMTS proteoglycanases, including ADAMTS4 and ADAMTS5, degrade CSPGs, representing potential targets for ameliorating axonal growth-inhibition by CSPG accumulation after CNS injury. We investigated the proteolysis of CSPGs in mice homozygous for Adamts4 or Adamts5 null alleles after spinal cord injury (SCI). ADAMTS-derived 50–60 kDa aggrecan and 50 kDa brevican fragments were observed in Adamts4−/−, Adamts5−/−, and wt mice but not in the sham-operated group. By contrast Adamts4−/− and Adamts5−/− mice were both protected from versican proteolysis with an ADAMTS-generated 70 kDa versican fragment predominately observed in WT mice. ADAMTS1, ADAMTS9, and ADAMTS15 were detected by Western blot in Adamts4−/− mice' spinal cords after SCI. Immunohistochemistry showed astrocyte accumulation at the injury site. These data indicate that aggrecan and brevican proteolysis is compensated in Adamts4−/− or Adamts5−/− mice by ADAMTS proteoglycanase family members but a threshold of versican proteolysis is sensitive to the loss of a single ADAMTS proteoglycanase during SCI. We show robust ADAMTS activity after SCI and exemplify the requirement for collective proteolysis for effective CSPG clearance during SCI.

Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition: A ROLE FOR ADAMTS5 PROTEASE-MEDIATED PROTEOLYSIS

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.

Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFβ3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.