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Table S1: Baseline patient demographics and disease characteristics. Table S2: mRNA/IHC AREG/EREG concordance table. Figure S1: AREG/EREG IHC scatterplot. Table S3: Prognostic effect of dichotomous AREG/EREG. Table S4: AREG/EREG and RECIST response rate. Table S5: AREG and EREG as individual continuous variables. Table S6: AREG/EREG predictive analyses adjusted for BRAF mutation, PTL and peritoneal metastases. Table S7: Exploratory analyses of different AREG/EREG cut points. Table S8: AREG/EREG IHC in biopsy and resection specimens.

Purpose:High tumor mRNA levels of the EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are associated with anti-EGFR agent response in metastatic colorectal cancer (mCRC). However, ligand RNA assays have not been adopted into routine practice due to issues with analytic precision and practicality. We investigated whether AREG/EREG IHC could predict benefit from the anti-EGFR agent panitumumab.Experimental Design:Artificial intelligence algorithms were developed to assess AREG/EREG IHC in 274 patients from the PICCOLO trial of irinotecan with or without panitumumab (Ir vs. IrPan) in RAS wild-type mCRC. The primary endpoint was progression-free survival (PFS). Secondary endpoints were RECIST response rate (RR) and overall survival (OS). Models were repeated adjusting separately for BRAF mutation status and primary tumor location (PTL).Results:High ligand expression was associated with significant PFS benefit from IrPan compared with Ir [8.0 vs. 3.2 months; HR, 0.54; 95% confidence interval (CI), 0.37–0.79; P = 0.001]; whereas low ligand expression was not (3.4 vs. 4.4 months; HR, 1.05; 95% CI, 0.74–1.49; P = 0.78). The ligand-treatment interaction was significant (Pinteraction = 0.02) and remained significant after adjustment for BRAF-mutation status and PTL. Likewise, RECIST RR was significantly improved in patients with high ligand expression (IrPan vs. Ir: 48% vs. 6%; P < 0.0001) but not those with low ligand expression (25% vs. 14%; P = 0.10; Pinteraction = 0.01). The effect on OS was similar but not statistically significant.Conclusions:AREG/EREG IHC identified patients who benefitted from the addition of panitumumab to irinotecan chemotherapy. IHC is a practicable assay that may be of use in routine practice.

Purpose: High tumor mRNA levels of the EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are associated with anti-EGFR agent response in metastatic colorectal cancer (mCRC). However, ligand RNA assays have not been adopted into routine practice due to issues with analytic precision and practicality. We investigated whether AREG/EREG IHC could predict benefit from the anti-EGFR agent panitumumab. Experimental Design: Artificial intelligence algorithms were developed to assess AREG/EREG IHC in 274 patients from the PICCOLO trial of irinotecan with or without panitumumab (Ir vs. IrPan) in RAS wild-type mCRC. The primary endpoint was progression-free survival (PFS). Secondary endpoints were RECIST response rate (RR) and overall survival (OS). Models were repeated adjusting separately for BRAF mutation status and primary tumor location (PTL). Results: High ligand expression was associated with significant PFS benefit from IrPan compared with Ir [8.0 vs. 3.2 months; HR, 0.54; 95% confidence interval (CI), 0.37–0.79; P = 0.001]; whereas low ligand expression was not (3.4 vs. 4.4 months; HR, 1.05; 95% CI, 0.74–1.49; P = 0.78). The ligand-treatment interaction was significant (Pinteraction = 0.02) and remained significant after adjustment for BRAF-mutation status and PTL. Likewise, RECIST RR was significantly improved in patients with high ligand expression (IrPan vs. Ir: 48% vs. 6%; P < 0.0001) but not those with low ligand expression (25% vs. 14%; P = 0.10; Pinteraction = 0.01). The effect on OS was similar but not statistically significant. Conclusions: AREG/EREG IHC identified patients who benefitted from the addition of panitumumab to irinotecan chemotherapy. IHC is a practicable assay that may be of use in routine practice.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of patients with deficient DNA mismatch repair (d-MMR) metastatic colorectal cancer (CRC), yet less than half of patients receive clinical benefit. We hypothesized that immune contexture including spatial distribution of cells expressing immune molecules in the tumor microenvironment may predict immunotherapy outcome. Methods: Primary CRC tissues from consecutive patients with d-MMR metastatic CRC (N=33) were treated with anti-PD-1 antibodies at Mayo Clinic Comprehensive Cancer Center (2015-2018). Tumors were stained for PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFβR2, MHC-I, CD14, B2M, DAPI and pan-cytokeratin in 5 compartments (overall tumor, tumor epithelia, tumor stroma, peritumor inside vs outside) by multiplex immunofluorescence with digital image analysis. Features computed within each image analysis region included positive cell density, fraction of positive cell types, intensity of positive cells, and spatial distribution between distinct cell types (distances measured using image analysis software). Prior to model fitting, feature selection was performed using regularized Cox regression with LASSO. Regularization parameter was chosen based on 5-fold cross validation. A Cox proportional hazards model was fitted to predict patient progression-free survival (PFS). Results: Among patients with d-MMR CRCs, 16/33 (48.4%) were female, 10 received first-line ICI therapy, and 23 had received > 1 prior chemotherapy regimen. Median age was 61.6 years (IQR of 49.4, 73.7). Eight patients (24.2%) harbored BRAFV600E, 9 (27.2%) had mutant KRAS, and median PFS was 22.2 months (95% CI: 11.8, NR) with 20 events. PD-L1 was expressed in tumor cells, CD68+ macrophages, and CD3+ T lymphocytes. PD-1 expression on CD8+ T lymphocytes was also observed. By univariate analysis, only cell-cell distance readouts achieved statistical significance. Multivariable feature selection identified the mean number of PD-1+ cells within 10 microns of a PD-L1+ cell in the overall tumor as the strongest predictor of anti-PD-1 efficacy, and was significantly associated with PFS (HR=0.87, 95% CI: 0.79-0.95, p< 0.001). In contrast, the ratio of PD-1+/PD-L1+ cells was not predictive of treatment efficacy (p= 0.47), thereby underscoring the importance of spatial distribution for PFS prediction. Conclusion: The mean number of PD-1+ cells in proximity to PD-L1+ cells in tumors was a predictive biomarker of anti-PD-1 efficacy. Confirmatory studies are warranted. Citation Format: Bahar Saberzadeh-Ardestani, Rondell P. Graham, Qian Shi, Eze Ahanonu, Sara McMahon, Crystal Williams, Antony Hubbard, Wenjun Zhang, Andrea Muranyi, Dongyao Yan, Kandavel Shanmugam, Frank A. Sinicrope. Prediction of anti-PD-1 efficacy based on immune marker densities and their spatial distribution in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6648.

This study leveraged a gene-protein assay to assess MYC and PTEN status at prostate cancer biopsy and examined the association with adverse outcomes after surgery. MYC gain and PTEN loss were simultaneously assessed by chromogenic in situ hybridization and immunohistochemistry, respectively, using 277 Grade Group 2 needle biopsies that were followed by prostatectomy. The maximal size of cribriform Gleason pattern 4 carcinoma (CRIB), the presence of intraductal carcinoma (IDC), and percentage of Gleason pattern 4 carcinoma at biopsy were also annotated. MYC gain or PTEN loss was present in 19% and 18% of biopsies, respectively, whereas both alterations were present in 9% of biopsies. Tumors with one or both alterations were significantly more likely to have non–organ-confined disease (NOCD) at radical prostatectomy. In logistic regression models, including clinical stage, tumor volume on biopsy, and presence of CRIB/IDC, cases with MYC gain and PTEN loss remained at higher risk for NOCD (odds ratio, 6.23; 95% CI, 1.74–24.55; P = 0.005). The area under the curve for a baseline model using CAPRA variables (age, prostate-specific antigen, percentage of core involvement, clinical stage) was increased from 0.68 to 0.69 with inclusion of CRIB/IDC status and to 0.75 with MYC/PTEN status. Dual MYC/PTEN status can be assessed in a single slide and is independently associated with increased risk of NOCD for Grade Group 2 biopsies.

203 Background: AREG and EREG are ligands of EGFR. AI assisted IHC evaluation of tumor AREG/EREG expression predicted benefit from anti-EGFR therapy in a retrospective analysis of the PICCOLO trial of second-line irinotecan ± panitumumab. Here, we sought to validate those findings through an analysis of patients who received anti-EGFR therapy during routine care for mCRC. Methods: Patients (pts) with available archival FFPE tumor tissue who received panitumumab or cetuximab ± chemotherapy at any time for treatment of mCRC at 8 UK cancer centers were eligible. Central RAS testing by next generation sequencing (NGS) was performed for pts where extended RAS testing had not been previously undertaken. RAS-mutant (mut) and RAS-unknown pts were excluded from the primary analysis population. AREG and EREG positive tumor cells were identified by IHC, performed locally at 6 of 8 sites. Pathologists annotated tumor areas on digital images of glass slides. AI algorithms calculated the percentage of tumor cells staining positive for AREG and EREG within tumor areas. More than 20% AREG and/or EREG tumor cell positivity was regarded as high biomarker expression, the optimal cut-point identified in PICCOLO. Study endpoints were progression-free survival (PFS), overall survival (OS), and locally reported response rate (RR) and disease control rate (DCR). Results: Of 541 pts recruited, 494 (91.3%) had adequate archival tissue for analysis. Central RAS testing was successfully performed in 255 of 393 (64.9%) pts without existing extended RAS results, leading to 45 exclusions, leaving 449 pts in the primary analysis population. 26 (5.8%) pts were BRAF-mut. 110 (24.5%) received concomitant FOLFOX and 304 (67.7%) FOLFIRI. After adjustment for additional prognostic factors, high AREG/EREG expression (n = 360; 80.2%) was associated with significantly prolonged PFS (HR 0.73; 95% CI, 0.56-0.95; p = 0.02), OS (HR 0.66 [0.50-0.86]; p = 0.002), and DCR (OR 1.92 [1.05-3.54]; p = 0.04), but not RR (unadjusted OR 1.39 [0.83-2.33]; p = 0.21). Median PFS in the high vs low biomarker groups was 8.5 vs 4.4 months; median OS 16.4 vs 8.9 months. The significant difference in OS (high vs low) was maintained in the subgroup with right-sided primary tumor location (n = 107; 23.8%) (HR 0.56 [0.37-0.86]; p = 0.007). Conclusions: High tumor AREG/EREG expression was associated with significantly prolonged PFS, OS and DCR among a cohort of pts treated with anti-EGFR therapy during routine care of mCRC. The prognostic effect observed validates the predictive effect of AREG/EREG seen in the PICCOLO trial, where AREG/EREG had no prognostic effect in patients receiving chemotherapy alone. A prospective biomarker-led trial is in set-up to support the use of AREG/EREG IHC in clinical practice.

204 Background: High CD3+ (all) and CD8+ (cytotoxic) T cell densities in the core (CT) and invasive margin (IM) of primary CRCs have been shown to be associated with superior prognosis at all stages of disease. Their predictive effect on benefit from adjuvant chemotherapy in early stage CRC has not been tested. Methods: FFPE samples from participants (pts) in the QUASAR trial (adjuvant fluorouracil/folinic acid vs observation in stage 2/3 CRC) were analysed for CD3 and CD8 immunohistochemistry (IHC). Pathologists annotated the core and peritumor areas on digital slide images. Artificial intelligence (AI) algorithms delineated the CT and IM, and calculated the densities (cells/mm2) of each marker in each region (CD3-CT, CD3-IM, CD8-CT, CD8-IM). Pts were randomly partitioned into test and validation sets (1:1). In the test set, each measure’s prognostic effect on recurrence-free interval (RFI) (primary endpoint), colorectal cancer mortality (CCM) and overall survival (OS) in each trial arm was assessed. Maximum likelihoods methods were used to develop optimal cut-points. Analyses were repeated in the validation set. Analysis of 425 pts in each set would give > 95% power (α = 0.05, 2-sided) to detect a twofold difference in recurrence risk. In predictive analyses, 2-year recurrence rate was the primary outcome; biomarker-treatment interactions were assessed. Results: Tumor tissue from 868 pts (797 [92%] stage 2; 531 [61%] colon) was analysed, with evaluable results for CD3-CT in 851 (98.0%), CD3-IM in 833 (96.0%), CD8-CT in 849 (97.0%) and CD8-IM in 820 (94.5%) pts. In the test set, optimal cut-points of 318, 798, 81 and 186 cells/mm2 were defined for CD3-CT, CD3-IM, CD8-CT and CD8-IM respectively. The recurrence rate in the high-risk group was twice that in the low-risk group for all measures (CD3-CT: rate ratio [RR] 2.00, [95%CI 1.33-2.94], p = 0.0008; CD3-IM: 2.38, [1.59-3.57], p < 0.00001; CD8-CT: 2.17, [1.59-3.57], p = 0.0001; CD8-IM: 2.13 [1.43-3.23], p = 0.0001), which was closely replicated in the validation set (CD3-CT: RR 1.96, [1.30-2.94], p = 0.002; CD3-IM: 1.79, [1.18-2.70], p = 0.005; CD8-CT: 1.72, [1.18-2.56], p = 0.005; CD8-IM: 1.72 [1.15-2.56], p = 0.008). In multivariate analyses, prognostic effects were similar in colon and rectal cancers, and in stage 2 and 3 disease. CD3/8 counts were not predictive of benefit from adjuvant chemotherapy, with similar efficacy in the high and low risk groups. Conclusions: AI-assisted CD3 and CD8 counts were strongly associated with tumor recurrence rates. With no biomarker-treatment interactions, proportional reductions in recurrence with chemotherapy were similar in high and low-risk disease. Hence, numbers of high-risk patients needed to treat to prevent one recurrence were about half the number for low-risk patients.

High tumor mRNA levels of the EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are associated with anti-EGFR agent response in metastatic colorectal cancer (mCRC). However, ligand RNA assays have not been adopted into routine practice due to issues with analytic precision and practicality. We investigated whether AREG/EREG IHC could predict benefit from the anti-EGFR agent panitumumab.Artificial intelligence algorithms were developed to assess AREG/EREG IHC in 274 patients from the PICCOLO trial of irinotecan with or without panitumumab (Ir vs. IrPan) inHigh ligand expression was associated with significant PFS benefit from IrPan compared with Ir [8.0 vs. 3.2 months; HR, 0.54; 95% confidence interval (CI), 0.37-0.79;AREG/EREG IHC identified patients who benefitted from the addition of panitumumab to irinotecan chemotherapy. IHC is a practicable assay that may be of use in routine practice.

PURPOSE: Understanding the molecular landscape of glioblastoma (GBM) is increasingly important in the age of targeted therapy. O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and EGFR amplification are markers that may play a role in prognostication, treatment, and/or clinical trial eligibility. Quantification of MGMT and EGFR protein expression may offer an alternative strategy towards understanding GBM. Here, we quantify baseline expression of MGMT and EGFR protein in newly diagnosed GBM samples using mass spectrometry. We correlate findings with MGMT methylation and EGFR amplification statuses and survival. METHODS: We retrospectively identified adult patients with newly diagnosed resected GBM. MGMT and EGFR protein expression were quantified using a selected reaction monitoring mass spectrometry assay. Protein levels were correlated with MGMT methylation and EGFR amplification and survival data. RESULTS: We found a statistically significant association between MGMT protein expression and promoter methylation status (p=0.02) as well as between EGFR protein expression and EGFR amplification (p

111 Background: High tumor mRNA levels of the EGFR ligands, AREG and EREG are associated with anti-EGFR agent response in patients (pts) with RAS-wt mCRC, regardless of tumor location. However, ligand RNA assays have not been adopted into routine clinical practice due to issues with analytical precision and practicality. Here we test whether AREG and EREG expression assessed by IHC can predict benefit from Pan. Methods: A retrospective biomarker study within the PICCOLO trial (NCT00389870; irinotecan [Ir] ± Pan in fluoropyrimidine-resistant RAS-wt mCRC). AREG and EREG positive tumor cells were assessed by IHC in all RAS-wt patients with available tumor tissue. Pathologists annotated tumor areas on digital images of glass slides. Artificial intelligence (AI) algorithms calculated the percentage of tumor cells staining positive for AREG and EREG within the tumor areas. More than 50% AREG and/or EREG tumor cell positivity was regarded as high ligand expression. The primary endpoint was progression-free survival (PFS) and secondary endpoints were RECIST response rate (RR) and overall survival (OS). Results: 274 RAS-wt pts had available tumor tissue. High ligand expression (n = 132) was associated with significant PFS benefit from IrPan compared with Ir (8.0 vs 3.2 months; HR 0.54 [0.37-0.79]; p = 0.001); whereas low ligand expression (n = 142) was not (3.4 vs 4.4 months; HR 1.05 [95% CI, 0.74-1.49]; p = 0.78). The ligand-treatment interaction was significant (p = 0.02) and independent of BRAF-mutation status and primary tumor location. Likewise RR was significantly improved in pts with high ligand expression (IrPan vs Ir: 48% vs 6%; risk ratio, 7.8 [2.90-20.69]; p < 0.0001) but not those with low ligand expression (IrPan vs Ir: 25% vs 14%; risk ratio, 1.8 [95% CI, 0.89-3.65]; p = 0.10) (interaction p = 0.01). Lesser effect was seen on OS. Conclusions: IHC assessment of AREG and EREG identified pts who did or did not benefit from Pan, as has been previously demonstrated through mRNA quantification. IHC represents a more practicable technique as it can be provided at the point of care and is associated with shorter turn-around times. AREG and EREG IHC may be of use in routine practice to identify patients who would benefit from anti-EGFR therapy and those for whom alternative treatment strategies should be explored.

Glioblastomas represent the most aggressive glioma grade and are associated with a poor patient prognosis. The current standard of care, consisting of surgery, radiation and chemotherapy, only results in a median survival of 14 months, underscoring the importance of developing effective new therapeutic strategies. Among the challenges in treating glioblastomas are primary resistance and the rapid emergence of recurrent disease, which can result from tumor cell-intrinsic mechanisms in addition to tumor microenvironment (TME)-mediated extrinsic resistance. Using a PDGF-B-driven proneural glioma mouse model, we assessed a panel of tyrosine kinase inhibitors with different selectivity profiles. We found that PLX3397, an inhibitor of colony stimulating factor-1 receptor (CSF-1R), blocks glioma progression, markedly suppresses tumor cell proliferation and reduces tumor grade. By contrast, the multi-targeted tyrosine kinase inhibitors dovitinib and vatalanib, which directly target tumor cells, exert minimal anti-tumoral effects in vivo, despite killing glioma cells in vitro, suggesting a TME-mediated resistance mechanism may be involved. Interestingly, PLX3397 interferes with tumor-mediated education of macrophages and consequently restores the sensitivity of glioma cells to tyrosine kinase inhibitors in vivo in preclinical combination trials. Our findings thus demonstrate that microenvironmental alteration by CSF-1R blockade renders tumor cells more susceptible to receptor tyrosine kinase inhibition in a preclinical glioblastoma model, which may have important translational relevance.

579 Background: Immune contexture shapes oncogenesis, tumor progression and response to therapy. Traditional techniques for studying the microenvironment, such as flow cytometry, are limited by low throughput. Recently, computational methods using transcriptomic data have enabled deconvolution of tumor immune landscape, particularly in colorectal cancer (CRC). However, the immune profiles of other GI and HN cancers remain unclear. We proposed that elucidating the immune compositions in those cancers would reveal biological insights and potentially therapeutic targets. Methods: 464 GI tumors with RNA-Seq data (∼200x106reads per tumor) from a commercial database were available for analysis. Tumors were categorized into CRC, gastroesophageal (GE), HN, and biliary cancer. A curated panel of 122 genes that discriminate between 28 immune subsets was identified. For each of these immune signatures, a database containing 1880 unselected tumors was used to define a distribution of expression. The study samples were then scored for their deviances within such distributions.In addition to immune deconvolution, each of the 464 tumors were assigned to one of the colorectal Consensus Molecular subtypes (CMS). Significant enrichment for immune subsets between locations and CMS was analyzed. Results: Unsupervised clustering revealed 2 clusters with distinct immune profiles, which largely separated CRC from HN/GE tumors (silhouette coefficient = 0.14). Eosinophils are more abundant in GI cancers than others. HN/GE tumors were characterized by abundant NK and Tregs (adj. p < 0.001), and were predominantly classified as the immune-active CMS1 (adj. p < 0.001). CRC was significantly associated with high eosinophils and fibroblasts (adj. p < 0.0001). Biliary tumors spanned both immune-type clusters, and were frequently classified as the stromal-rich CMS4 (adj. p < 0.0001). Immature dendritic cells were sparse in GI tumors, especially GE. Conclusions: Upper and lower GI tumors have distinct immune contextures, which may differentially impact the efficacy of immunotherapy. Comprehensive immune profiling may facilitate the identification of targetable immune subsets.

Bovine lactoferricin (LfcinB) is a multifunctional peptide derived from bovine lactoferrin that demonstrates antibacterial, antifungal, antiviral, antitumor, and immunomodulatory activities. Recently, studies have focused on the anti-catabolic and anti-inflammatory potential of LfcinB. LfcinB is able to modulate the effects cytokines such as IL-1 and fibroblast growth factor 2 as well as promote specific cartilage anabolic factors. These properties are particularly important in maintaining cartilage homeostasis and preventing a catabolic state, which leads to clinical pathology. This review focuses on the recent literature elucidating the role of LfcinB in preventing cartilage degradation.

Bone-morphogenetic protein-7 (BMP7) is a well-known anabolic and anti-catabolic growth factor on intervertebral disc (IVD) matrix and cell homeostasis. Similarly, Lactoferricin B (LfcinB) has recently been shown to have pro-anabolic, anti-catabolic, anti-oxidative and/or anti-inflammatory effects in bovine disc cells in vitro. In this study, we investigated the potential benefits of using combined peptide therapy with LfcinB and BMP7 for intervertebral disc matrix repair and to understand cellular and signaling mechanisms controlled by these factors. We studied the effects of BMP7 and LfcinB as individual treatments and combined therapy on bovine nucleus pulposus (NP) cells by assessing proteoglycan (PG) accumulation and synthesis, and the gene expression of matrix protein aggrecan and transcription factor SOX-9. We also analyzed the role of Noggin, a BMP antagonist, in IVD tissue and examined its effect after stimulation with LfcinB. To understand the molecular mechanisms by which LfcinB synergizes with BMP7, we investigated the ERK-SP1 axis as a downstream intracellular signaling regulator involved in BMP7 and LfcinB-mediated activities. Treatment of bovine NP cells cultured in alginate with LfcinB plus BMP7 synergistically stimulates PG synthesis and accumulation in part by upregulation of aggrecan gene expression. The synergism results from LfcinB-mediated activation of Sp1 and SMAD signaling pathways by (i) phosphorylation of SMAD 1/5/8; (ii) downregulation of SMAD inhibitory factors [i.e., noggin and SMAD6 (inhibitory SMAD)]; and (iii) upregulation of SMAD4 (universal co-SMAD). These data indicate that LfcinB-suppression of Noggin may eliminate the negative feedback of BMP7, thereby maximizing biological activity of BMP7 and ultimately shifting homeostasis to a pro-anabolic state in disc cells. We propose that combination growth factor therapy using BMP7 and LfcinB may be beneficial for treatment of disc degeneration.

Bovine lactoferricin (LfcinB) is a multi-functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin-1 β) IL-1β and FGF-2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL-1β and FGF-2 on the expression of cartilage-degrading enzymes (MMP-1, MMP-3, and MMP-13), destructive cytokines (IL-1β and IL-6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL-4 and IL-10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti-inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL-1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti-catabolic and anti-inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future.

Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes.

Existing literature demonstrates that fibroblast growth factor-2 (FGF-2) exerts opposing, contradictory biological effects on cartilage homeostasis in different species. In human articular cartilage, FGF-2 plays a catabolic and anti-anabolic role in cartilage homeostasis, driving homeostasis toward degeneration and osteoarthritis (OA). In murine joints, however, FGF-2 has been identified as an anabolic mediator as ablation of the FGF-2 gene demonstrated increased susceptibility to OA. There have been no previous studies specifically addressing species-specific differences in FGF-2-mediated biological effects. In this study, we provide a mechanistic understanding by which FGF-2 exerts contradictory biological effects in human versus murine tissues. Using human articular cartilage (ex vivo) and a medial meniscal destabilization (DMM) animal model (in vivo), species-specific expression patterns of FGFR receptors (FGFRs) are elucidated between human and murine articular cartilage. In the murine OA model followed by intra-articular injection of FGF-2, we further correlate FGFR profiles to changes in behavioral pain perception, proteoglycan content in articular cartilage, and production of inflammatory (CD11b) and angiogenic (VEGF) mediators in synovium lining cells. Our results suggest that the fundamental differences in cellular responses between human and murine tissues may be secondary to distinctive expression patterns of FGFRs that eventually determine biological outcomes in the presence of FGF-2. The complex interplay of FGFRs and the downstream signaling cascades induced by FGF-2 in human cartilage should add caution to the use of this particular growth factor for biological therapy in the future.

Glioblastoma (GBM) is an aggressive primary brain tumor with median progression-free survival (PFS) of 7 months and median overall survival (OS) of 15 months. Standard treatment at diagnosis consists of surgery, radiation and temozolomide (TMZ). O-6-methylguanine DNA methyltransferase (MGMT) modulates TMZ effect and MGMT promoter methylation status is used to predict outcomes and guide treatment decisions. However, standard testing is limited by poor inter-assay reproducibility and a weak correlation between methylation status and MGMT expression. We quantitated MGMT protein by mass spectrometry in 32 GBM patients to assess its relationship to PFS and OS after upfront TMZ treatment. We obtained archived tumor samples from newly diagnosed GBM patients prior to treatment with surgery, radiation and TMZ. Tumor cells were microdissected and solubilized, and MGMT protein was quantitated using mass spectrometry. PFS (determined by RANO) and OS were assessed using the Kaplan-Meier method and log-rank test. Of the 32 patients (66% male; median age: 63 years),15 expressed MGMT protein. While methylation status agreed with absence of MGMT protein in 8/9 cases, 9/23 samples with unmethylated MGMT had undetectable MGMT protein, indicating poor agreement between methylation status and protein expression. Patients with MGMT levels below 150 amol/ug of total protein had longer PFS than those with higher MGMT levels (HR: 0.51; p=0.0399; median PFS: 303 vs. 260 days). Similarly, MGMT level below 150 amol/ug was associated with longer OS (HR: 0.49; p=0.0311; median OS: 659 vs. 534 days). Discordance between MGMT methylation status and protein expression was found in 31% of GBM samples. MGMT protein expression below 150 amol/ug correlated with longer PFS and OS in TMZ-treated patients. Mass spectrometry-based MGMT quantitation provides a direct readout of protein expression and may be more reliable than methylation testing.

Dynamic modification involving small ubiquitin-like modifier (SUMO) has emerged as a new mechanism of protein regulation in mammalian biology. Sumoylation is an ATP-dependent, reversible post-translational modification which occurs under both basal and stressful cellular conditions. Sumoylation profoundly influences protein functions and pertinent biological processes. For example, sumoylation modulates multiple components in the NFkappaB pathway and exerts an anti-inflammatory effect. Likewise, sumoylation of peroxisome proliferator-activated receptor gamma (PPARgamma) augments its anti-inflammatory activity. Current evidence suggests a role of sumoylation for resistance to apoptosis in synovial fibroblasts. Dynamic SUMO regulation controls the biological outcomes initiated by various growth factors involved in cartilage homeostasis, including basic fibroblast growth factors (bFGF or FGF-2), transforming growth factor-beta (TGF-beta) and insulin-like growth factor-1 (IGF-1). The impact of these growth factors on cartilage are through sumoylation-dependent control of the transcription factors (e.g., Smad, Elk-1, HIF-1) that are key regulators of matrix components (e.g., aggrecan, collagen) or cartilage-degrading enzymes (e.g., MMPs, aggrecanases). Thus, SUMO modification appears to profoundly affect chondrocyte and synovial fibroblast biology, including cell survival, inflammatory responses, matrix metabolism and hypoxic responses. More recently, evidence suggests that, in addition to their nuclear roles, the SUMO pathways play crucial roles in mitochondrial activity, cellular senescence, and autophagy. With an increasing number of reports linking SUMO to human diseases like arthritis, it is probable that novel and equally important functions of the sumoylation pathway will be elucidated in the near future.

Background: Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands accounting for 10% of salivary gland malignancies and 1% of head and neck cancers. About 1,200 new cases are diagnosed annually in the United States. ACC is typically chemoresistant and clinical trials of multiple targeted agents found few responders. Studies of ACC oncogenesis have described frequent fusion of MYB-NFIB genes and other infrequent genomic mutations. ACC has not been proteomically characterized. We hypothesized that proteogenomic analysis of ACC tumor tissues would identify clinically relevant molecular differences between ACC and other tumor types. The objective of this study was to identify altered disease pathways and potential drug targets in ACC. Methods: Clinical tumor samples of ACC and squamous cell carcinoma (SCC) of the head and neck were analyzed using the GPS Cancer diagnostic test, which includes whole genome and transcriptome sequencing, and proteomic expression analysis using mass spectrometry. To quantify proteins, tumor areas of formalin-fixed paraffin-embedded tissue sections were marked by a pathologist, microdissected and solubilized. The resulting lysate was analyzed with mass spectrometry to quantitate 30 clinically relevant proteins. Samples were further analyzed with a global proteomics platform intended to discover targetable protein biomarkers. Proteins that were overexpressed or underexpressed (defined as a 1.5-fold difference between ACC and SCC) were subjected to pathway analysis to identify perturbed pathways and potential drug targets. Finally, the mRNA expression in ACC tumors was compared with RNA-seq data from various solid tumor types using the k-nearest neighbors algorithm. Results: In 14 tumor samples, unsupervised hierarchical clustering analysis of 4,002 proteins revealed a clear separation between ACC (n=8) and SCC (n=6) tissues. Four of 8 ACC samples harbored an MYB-NFIB fusion and single samples had fusions of MYBL-NFIB or AHI1-NFIB. Mutational burden in ACC and SCC samples was 1.35 and 3.53 mutations per megabase, respectively. Pathway analysis found enrichment of ACC genes in essential cell cycle processes. CDK6 protein expression was 4-fold higher in ACC samples than SCC samples by mass spectrometry (p=0.0036) and 3-fold higher at the mRNA level. Expression of p16 protein was 3-fold lower in ACC than in SCC (p=0.0289) and corresponding p16 mRNA levels were 17-fold lower in ACC than SCC. All ACC samples harbored intact retinoblastoma (RB1) gene. Expression of mRNA in 15 ACC samples was compared with that of 25 different tumor types from TCGA and the authors' clinical laboratory; breast cancer was found to be the nearest neighbor to ACC. Conclusions: Proteogenomic analysis revealed CDK6 overexpression in ACC. The combination of CDK6 overexpression, p16 underexpression and RB1 proficiency suggests that ACC tumors may respond to treatment with CDK6 inhibitors. Citation Format: Sheeno P. Thyparambil, Yeoun Jin Kim, Andrew G. Chambers, Dongyao Yan, Shankar Sellappan, Andrew J. Sedgewick, Yulia Newton, J Zachary Sanborn, Charles J. Vaske, Stephen C. Benz, Fabiola Cecchi, Hyunseok Kang, Todd A. Hembrough. Potential drug targets for adenoid cystic carcinoma elucidated by proteogenomic analysis [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 778.

Background: Chemotherapy represents the cornerstone of treatment for patients with advanced sarcomas. Anthracyclines are the first line of treatment; however, other agents such as gemcitabine, topotecan, taxanes and temozolomide have shown clinical activity in sarcoma patients. While no biomarker for chemotherapy has been approved for clinical use, a growing body of literature has identified tumor molecular characteristics predictive of response or resistance to chemotherapy. These include expression of the proteins ERCC1 (platinum), TUBB3 (taxane), TOP2A (anthracyclines), TOP1 (irinotecan, topotecan), hENT1/RRM1 (gemcitabine) and MGMT (temozolomide). Our goal was to assess the landscape of chemotherapy biomarkers in several sarcoma subtypes to identify potentially beneficial treatment regimens. Methods: Formalin-fixed, paraffin-embedded tumor tissues from clinical samples of sarcoma representing 5 subtypes were microdissected and assayed with the GPS Cancer molecular test. The test combines whole-genome sequencing, RNA-seq, and proteomic expression analysis of 30 biomarker proteins using mass spectrometry. Results: Seventy-five sarcoma samples were characterized (Table 1). Expression of protein biomarkers varied by sarcoma subtype. Of interest, approximately two-thirds of leiomyosarcomas lacked resistance markers for platinum (ERCC1) and taxanes (TUBB3). Of osteosarcomas, 93% expressed the response marker for gemcitabine (hENT1), while only 14% expressed the protein marker of gemcitabine resistance (RRM1). The vast majority of Ewing's sarcomas expressed MGMT, a marker of temozolomide resistance. Conclusions: In clinical sarcoma samples, proteomic analysis can identify distinct patterns of expression that are predictive of response or resistance to chemotherapies. Additional analysis is in progress; correlation between expression of chemotherapeutic biomarkers and clinical outcomes in a subset of sarcoma patients will be presented. Table 1. Proportions of sarcoma patients (N=75) with tumor expression of chemotherapeutic biomarker proteinsMarkers of response (agent)Markers of resistance (agent)Sarcoma subtypeTOP1(topotecan, irinotecan)TOP2A(doxorubicin)hENT1(gemcitabine)ERCC1(platinum)TUBB3(taxane)RRM1(gemcitabine)MGMT(temozolomide)Leiomyosarcoma (n=25)4133228321658Liposarcoma (n=13)1504654621667Ewing's sarcoma (n=12)3383383504288Osteosarcoma (n=14)779346571467Rhabdomyosarcoma (n=11)36278264456488Tumor expression is defined as quantitated protein above the threshold determined to be indicative of clinical response/resistance. The thresholds (in attomoles per microgram of total tumor protein) are as follows: TOP1: 1340, TOP2A: 1570, hENT1: 100, ERCC1: 75, TUBB3: 850, RRM1: 700, MGMT: 200. Citation Format: Sheeno P. Thyparambil, Shankar Sellappan, Dongyao Yan, Fabiola Cecchi, Antoine Italiano, Todd A. Hembrough. The molecular landscape of sarcoma can inform selection of personalized chemotherapy [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 1628.

6053Background: Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands accounting for 10% of salivary gland cancers and 1% of head and neck cancers. ACC rarely responds to chemotherapy...

8574Background: Even with standard platinum doublet therapy, most patients with small-cell lung cancer (SCLC) survive < 1 year. For those with refractory or relapsed disease, second-line, single-ag...

708 Background: 5-fluorouracil (5-FU) is a common adjuvant treatment for stage III and high-risk stage II colorectal cancer (CRC). However, about 20% of patients relapse within 48 months of treatment with 5-FU, even when combined with oxaliplatin. To improve patient selection, tumor biomarkers that predict sensitivity to 5-FU have been proposed. These include proteins involved in 5-FU activation or metabolism such as uridine-cytidine kinase 2 (UCK2) and thymidylate synthase (TYMS). We used multiplexed mass spectrometry to evaluate the utility these biomarkers in the archived tumor samples of patients with stage II/III CRC. Methods: Tumor samples were from 143 patients with stage II/III CRC who received adjuvant 5-FU, folinic acid, and oxaliplatin during 2000-2014; 83% of patients received 12 cycles and the others received ≤ 11 cycles. Tumor cells were microdissected and solubilized, and 67 candidate biomarkers were quantitated using mass spectrometry. Overall survival (OS) and relapse-free survival (RFS) were assessed using the Kaplan-Meier method and log-rank test. Protein expression by tumor stage, lymph node (LN) status, tumor sidedness was compared using the Student’s t-test. Results: Of 143 patients, 45 had recurrence and 98 patients did not. UCK2 was detected in all samples, ranging from 187 to 1606 attomoles per microgram of total protein (amol/µg). Patients with UCK2 expression above 335 amol/μg (n = 109) had significantly longer OS than patients with lower expression (n = 34; HR: 0.42; p= 0.009). There was no significant difference in RFS (HR: 0.6; p= 0.088). UCK2 expression did not differ by disease stage, LN metastasis status, or tumor sidedness. TYMS expression was not associated with survival in this cohort. Analysis of other biomarkers associated with response to 5-FU and platinum is in progress. Conclusions: In stage II/III CRC, UCK2 expression above 335 amol/μg identifies a subgroup of 5-FU-treated CRC patients with longer survival, suggesting that quantitated UCK2 has potential for use in selecting patients for treatment. These findings warrant validation in larger cohorts.

Bovine lactoferricin (LfcinB), a multifunctional peptide, was recently demonstrated to be anti-catabolic and anti-inflammatory in human articular cartilage. LfcinB blocks IL-1-mediated proteoglycan depletion, matrix-degrading enzyme expression, and pro-inflammatory mediator induction. LfcinB selectively activates ERK1/2, p38 (but not JNK), and Akt signaling. However, the relationship between these pathways and LfcinB target genes has never been explored. In this study, we uncovered the remarkable ability of LfcinB in the induction of an anti-inflammatory cytokine, IL-11. LfcinB binds to cell surface heparan sulfate to initiate ERK1/2 signaling and activate AP-1 complexes composed of c-Fos and JunD, which transactivate the IL-11 gene. The induced IL-11 functions as an anti-inflammatory and chondroprotective cytokine in articular chondrocytes. Our data show that IL-11 directly attenuates IL-1-mediated catabolic and inflammatory processes ex vivo and in vitro. Moreover, IL-11 activates STAT3 signaling pathway to critically up-regulate TIMP-1 expression, as a consecutive secondary cellular response after IL-11 induction by LfcinB-ERK-AP-1 axis in human adult articular chondrocytes. The pathological relevance of IL-11 signaling to osteoarthritis is evidenced by significant down-regulation of its cognate receptor expression in osteoarthritic chondrocytes. Together, our results suggest a two-step mechanism, whereby LfcinB induces TIMP-1 through an IL-11-dependent pathway involving transcription factor AP-1 and STAT3.

The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future.

Osteoarthritis afflicts millions of individuals across the world resulting in impaired quality of life and increased health costs. To understand this disease, physicians have been studying risk factors, such as genetic predisposition, aging, obesity, and joint malalignment; however have been unable to conclusively determine the direct etiology. Current treatment options are short-term or ineffective and fail to address pathophysiological and biochemical mechanisms involved with cartilage degeneration and the induction of pain in arthritic joints. OA pain involves a complex integration of sensory, affective, and cognitive processes that integrate a variety of abnormal cellular mechanisms at both peripheral and central (spinal and supraspinal) levels of the nervous system Through studies examined by investigators, the role of growth factors and cytokines has increasingly become more relevant in examining their effects on articular cartilage homeostasis and the development of osteoarthritis and osteoarthritis-associated pain. Catabolic factors involved in both cartilage degradation in vitro and nociceptive stimulation include IL-1, IL-6, TNF-α, PGE2, FGF-2 and PKCδ, and pharmacologic inhibitors to these mediators, as well as compounds such as RSV and LfcinB, may potentially be used as biological treatments in the future. This review explores several biochemical mediators involved in OA and pain, and provides a framework for the understanding of potential biologic therapies in the treatment of degenerative joint disease in the future.

Osteoarthritis (OA) and degenerative disc disease (DDD) are similar diseases involving the breakdown of cartilage tissue, and a better understanding of the underlying biochemical processes involved in cartilage degeneration may allow for the development of novel biologic therapies aimed at slowing the disease process. Three members of the fibroblast growth factor (FGF) family, FGF-2, FGF-18, and FGF-8, have been implicated as contributing factors in cartilage homeostasis. The role of FGF-2 is controversial in both articular and intervertebral disc (IVD) cartilage as it has been associated with species- and age-dependent anabolic or catabolic events. Recent evidence suggests that FGF-2 selectively activates FGF receptor 1 (FGFR1) to exert catabolic effects in human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme production, inhibition of extracellular matrix (ECM) accumulation and proteoglycan synthesis, and clustering of cells characteristic of arthritic states. FGF-18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating the FGFR3 pathway, inducing ECM formation and chondrogenic cell differentiation, and inhibiting cell proliferation. These changes result in dispersed chondrocytes or disc cells surrounded by abundant matrix. The role of FGF-8 has recently been identified as a catabolic mediator in rat and rabbit articular cartilage, but its precise biological impact on human adult articular cartilage or IVD tissue remains unknown. The available evidence reveals the promise of FGF-2/FGFR1 antagonists, FGF-18/FGFR3 agonists, and FGF-8 antagonists (i.e., anti-FGF-8 antibody) as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future.

Osteoarthritis, a debilitating degenerative joint disease predominantly found in elderly individuals, has become the principal source of physical disability resulting in increased health care costs and impaired quality of life in the United States. The pathogenesis of osteoarthritis (OA) involves the progressive deterioration of cartilage tissue, but many of the underlying biochemical and pathophysiological mechanisms involved in cartilage degradation and the induction of pain in this process remain largely unknown. Recent literature has focused on understanding many of these processes, with the intention of developing novel therapies aimed at slowing and/or reversing cartilage degradation and inducing symptomatic relief. This chapter provides an overview of several biochemical mediators involved in OA, with an emphasis on reviewing pertinent factors mediating cartilage breakdown and the induction of pain in degenerative conditions.

The natural phytoestrogen resveratrol (RSV) may have therapeutic potential for arthritic conditions. RSV is chondroprotective for articular cartilage in rabbit models for arthritis, but its biological effects on human articular cartilage and chondrosarcoma cells are unknown. Effects of RSV on human articular cartilage homeostasis were studied by assessing production of matrix-degrading enzymes (MMP-13, ADAMTS4, and ADAMTS5), as well as proteoglycan production and synthesis. The counteractions of RSV against catabolic factors (e.g., FGF-2 or IL-1β) were examined by in vitro and ex vivo using monolayer, three-dimensional alginate beads and cartilage explants cultures, respectively. RSV improves cell viability of articular chondrocytes and effectively antagonizes cartilage-degrading protease production that was initiated by catabolic and/or anti-anabolic cytokines in human articular chondrocytes. RSV significantly also enhances BMP7-promoted proteoglycan synthesis as assessed by (35) S-sulfate incorporation. Protein-DNA interaction arrays suggest that RSV inhibits the activation of transcription factors involved in inflammation and cartilage catabolic signaling pathways, including direct downstream regulators of MAPK (e.g., AP-1, PEA3) and NFκB. RSV selectively compromises survival of human chondrosarcoma cells, but not primary articular chondrocytes, revealing cell-specific activity of RSV on non-tumorigenic versus tumor-derived cells. We propose that RSV exerts its chondroprotective functions, in part, by deactivating p53-induced apoptosis in human primary chondrocytes, but not human chondrosarcoma. Our findings suggest that RSV has potential as a unique biologic treatment for both prevention and treatment of cartilage degenerative diseases.

Objective Protein kinase Cδ (PKCδ) activation has been shown to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. The aim of this study was to assess the role of the PKC pathways, specifically PKCδ, in intervertebral disc tissue homeostasis. Methods Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiologic role of the PKCδ pathway by examining 1) proteoglycan deposition, 2) matrix-degrading enzyme production and activity, 3) downstream signaling pathways regulated by PKCδ, and 4) the effect on in vivo models of disc degeneration in genetically engineered PKCδ-knockout mice. Results Studies of pathway-specific inhibitors revealed a vital role of the PKCδ/MAPK (ERK, p38, JNK) axis and NF-κB in disc homeostasis. Accordingly, in an in vivo model of disc injury, PKCδ-knockout mice were markedly resistant to disc degeneration. Conclusion Suppression of the PKCδ pathway may be beneficial in the prevention and/or treatment of disc degeneration. The results of this study provide evidence for a potential therapeutic role of pathway-specific inhibitors of the PKCδ cascade in the future.

Lactoferricin (LfcinB) antagonizes biological effects mediated by angiogenic and catabolic growth factors, in addition to pro-inflammatory cytokines and chemokines in human endothelial cells and tumor cells. However, the effect of LfcinB on intervertebral disc (IVD) cell metabolism has not yet been investigated. Using bovine nucleus pulposus (NP) cells, we analyzed the effect of LfcinB on proteoglycan (PG) accumulation, PG synthesis, and anabolic gene expression. We assessed expression of genes for matrix-degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family), as well as their endogenous inhibitors, tissue inhibitor of metalloproteases (TIMPs). In order to understand the specific molecular mechanisms by which LfcinB exerts its biological effects, we investigated intracellular signaling pathways in NP cells. LfcinB increased PG accumulation mainly via PG synthesis in a dose-dependent manner. Simultaneously, LfcinB dose-dependently downregulated catabolic enzymes. LfcinB's anti-catabolic effects were further demonstrated by a dose-dependent increase in multiple TIMP family members. Our results demonstrate that ERK and/or p38 mitogen-activated protein kinase pathways are the key signaling cascades that exert the biological effects of LfcinB in NP cells, regulating transcription of aggrecan, SOX-9, TIMP-1, TIMP-2, TIMP-3, and iNOS. Our results suggest that LfcinB has anabolic and potent anti-catabolic biological effects on bovine IVD cells that may have considerable promise in the treatment of disc degeneration in the future.

Hee-Jeong Im,1–4 Andrew D Sharrocks,5 Xia Lin,6 Dongyao Yan,1 Jaesung Kim,1 Andre J van Wijnen,7 Robert A Hipskind81Departments of Biochemistry, 2Internal Medicine, 3Section of Rheumatology, Orthopedic Surgery, 4Rush University Medical Center, and Department of Bioengineering; University of Illinois at Chicago, IL USA; 5Faculty of Life Sciences, University of Manchester, Oxford Rd, Manchester, UK; 6MichaelD DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA; 7Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; 8Institute De Genetique Moleculaire de Montpellier, FranceAbstract: Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and release of basic fibroblast growth factor (bFGF) are principal aspects of the pathology of osteoarthritis (OA). ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO) serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation-dependent transactivation capacity of Elk-1. This attenuates transcription of its downstream target gene MMP-13 to maintain the integrity of cartilage ECM homeostasis.Keywords: osteoarthritis, MMP-13, bFGF, SUMO, Elk-1

INTRODUCTION: Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA. METHODS: Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity. RESULTS: Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes. CONCLUSIONS: FGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.

Introduction: Degeneration of the interverterbral disk is as a cause of low-back pain is increasing. To gain insight into relationships between biological processes, structural alterations and behavioral pain, we created an animal model in rats. Methods: Disk degeneration was induced by removal of the nucleus pulposus (NP) from the lumbar disks (L4/L5 and L5/L6) of Sprague Dawley rats using a 0.5-mm-diameter microsurgical drill. The degree of primary hyperalgesia was assessed by using an algometer to measure pain upon external pressure on injured lumbar disks. Biochemical and histological assessments and radiographs of injured disks were used for evaluation. We investigated therapeutic modulation of chronic pain by administering pharmaceutical drugs in this animal model. Results: After removal of the NP, pressure hyperalgesia developed over the lower back. Nine weeks after surgery we observed damaged or degenerated disks with proteoglycan loss and narrowing of disk height. These biological and structural changes in disks were closely related to the sustained pain hyperalgesia. A high dose of morphine (6.7 mg/kg) resulted in effective pain relief. However, high doses of pregabalin (20 mg/kg), a drug that has been used for treatment of chronic neuropathic pain, as well as the anti-inflammatory drugs celecoxib (50 mg/kg; a selective inhibitor of cyclooxygenase 2 (COX-2)) and ketorolac (20 mg/kg; an inhibitor of COX-1 and COX-2), did not have significant antihyperalgesic effects in our disk injury animal model. Conclusions: Although similarities in gene expression profiles suggest potential overlap in chronic pain pathways linked to disk injury or neuropathy, drug-testing results suggest that pain pathways linked to these two chronic pain conditions are mechanistically distinct. Our findings provide a foundation for future research on new therapeutic interventions that can lead to improvements in the treatment of patients with back pain due to disk degeneration.