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Bevacizumab in neoadjuvant therapy provides a new hope of improved survival for patients with triple-negative breast cancer (TNBC) by targeting vascular endothelial growth factor in combination with chemotherapy, but curative effect is limited by bevacizumab’s continuous use while mechanisms remain incompletely understood. More and more researches reported that tumor-associated macrophages mediate resistance to chemotherapy and radiotherapy in various tumors. Here we developed a TNBC model resistant to bevacizumab under bevacizumab continuous administration. It was found that proportion of a specific subset of tumor-associated macrophages characterized as M2b (CD11b+ CD86high IL10high) increased and responsible for acquired resistance to bevacizumab. Then, we showed that RAW264.7 macrophages could be polarized to M2b subtype on simultaneous exposure to bevacizumab and TLR4 ligands as occurs in the context of continuous bevacizumab treatment. Concordantly, in TLR4-deleted C57BL/10ScNJNju (TLR4lps–del) mut/mut mice with bevacizumab treatment model, it was verified that the M2b macrophage could be induced by Fc gamma receptor-TLR4 cross-talk. In MDA-MB-231-resistant tumor-bearing mice, the content of TNFα in serum kept going up consistent with CCL1, a chemokine of M2b macrophage. In vitro neutralizing tumor necrosis factor α (TNFα) could inhibit the tumor progression caused by M2b culture medium and tumor IDO1 expression. Therefore, we thought that TNFα is a key tumor-promoting effector molecule secreted by M2b macrophage. Accordingly, the curative effect of bevacizumab was proved to be significantly improved by neutralizing TNFα with anti-TNFα nanobody. This study is expected to provide theoretical and clinical evidence elucidating the drug resistance in patients receiving bevacizumab.

Summary Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14−/−) and man causes dwarfism, osteopenia, arthritis, and lipodystrophy, abnormalities ascribed to defective collagen turnover. We have previously shown non-proteolytic functions of MT1-MMP mediated by its cytoplasmic tail, where the unique tyrosine (Y573) controls intracellular signaling. The Y573D mutation blocks TIMP-2/MT1-MMP-induced Erk1/2 and Akt signaling without affecting proteolytic activity. Here, we report that a mouse with the MT1-MMP Y573D mutation (Mmp14Y573D/Y573D) shows abnormalities similar to but also different from those of Mmp14−/− mice. Skeletal stem cells (SSC) of Mmp14Y573D/Y573D mice show defective differentiation consistent with the mouse phenotype, which is rescued by wild-type SSC transplant. These results provide the first in vivo demonstration that MT1-MMP modulates bone, cartilage, and fat homeostasis by controlling SSC differentiation through a mechanism independent of proteolysis., Graphical Abstract, Highlights • The proteinase MT1-MMP controls signaling by its non-proteolytic cytoplasmic tail • The MT1-MMP cytoplasmic tail regulates skeletal stem cell differentiation • MT1-MMP modulates bone, joint cartilage, and fat homeostasis by its cytoplasmic tail, Pathophysiology; Cell Biology

SUMMARYMembrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix (ECM) remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14−/−) and man causes dwarfism, osteopenia, arthritis and lipodystrophy, abnormalities ascribed to defective collagen turnover. We have previously shown non-proteolytic functions of MT1-MMP mediated by its cytoplasmic tail, where the unique tyrosine (Y573) controls intracellular signaling. The Y573D mutation blocks TIMP2/MT1-MMP-induced Erk1/2 and Akt signaling without affecting proteolytic activity. Here we report that a mouse with the MT1-MMP Y573D mutation (Mmp14Y573D/Y573D) shows abnormalities similar to, but also different from those ofMmp14−/−mice. Skeletal stem cells (SSC) ofMmp14Y573D/Y573Dmice show defective differentiation consistent with the mouse phenotype, which is rescued by wild-type SSC transplant. These results provide the firstin vivodemonstration that MT1-MMP modulates bone, cartilage and fat homeostasis by controlling SSC differentiation through a mechanism independent of proteolysis.

Osteoarthritis (OA) is characterized by progressive loss of articular cartilage accompanied by the new bone formation and, often, a synovial proliferation that culminates in pain, loss of joint function, and disability. However, the cellular and molecular mechanisms of OA progression and the relative contributions of cartilage, bone, and synovium remain unclear. We recently found that the extracellular matrix (ECM) protein periostin (Postn, or osteoblast-specific factor, OSF-2) is expressed at high levels in human OA cartilage. Multiple groups have also reported elevated expression of Postn in several rodent models of OA. We have previously reported that in vitro Postn promotes collagen and proteoglycan degradation in human chondrocytes through AKT/β-catenin signaling and downstream activation of MMP-13 and ADAMTS4 expression. Here we show that Postn induces collagen and proteoglycan degradation in cartilage by signaling through discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase. The genetic deficiency or pharmacological inhibition of DDR1 in mouse chondrocytes blocks Postn-induced MMP-13 expression. These data show that Postn is signaling though DDR1 is mechanistically involved in OA pathophysiology. Specific inhibitors of DDR1 may provide therapeutic opportunities to treat OA.

Aims Triple-negative breast cancer (TNBC) is not sensitive to current endocrine treatments, so new treatment strategies need to be explored. Based on previous antitumour studies on anti-TNFα nanobody, we designed a novel fusion nanobody to enhance antitumour activity of the anti-TNFα nanobody in TNBC. Main methods The RGD4C contains RGD sequence, which is the smallest recognition unit binding to the αvβ3 receptor on tumour cell membranes and involved in tumour cell adhesion, proliferation, and metastasis. RGD4C was fused to anti-TNFα nanobody to investigate the antitumour activity in vitro and in vivo. Key findings The antitumour effects of fusion nanobody V-L-R-H could effectively bind to αvβ3 and inhibit cell migration and proliferation of MDA-MB-231, which had satisfying purification efficiency and approving antigen or receptor binding activity. V-L-R-H could inhibit the TNFα-mediated PI3K/AKT/NF-κB signal pathway and integrin αvβ3 correlative FAK focal adhesion signal pathway. Mouse xenograft tumour experiments showed that the V-L-R-H could inhibit tumour proliferation and metastasis; reduce the TNFα, HIFα, Ki67, and CD31 concentrations in tumour; and inhibit the process of epithelial-mesenchymal transition. Significance The fusion nanobody enhanced antitumour activity of the anti-TNFα nanobody on TNBC. It provided a reference for the design of dual functional fusion proteins and development of tumour treatment strategies of antagonistic TNFα and αvβ3, and a new therapeutic strategy and research direction for the treatment of TNBC.

Background: To investigate the expression of vascular adhesion protein-1 (VAP-1) in joint tissues and serum in symptomatic knee osteoarthritis (SKOA) patients and examine whether VAP-1 levels predict increased risk of disease severity in a cross-sectional study. Methods: Baseline VAP-1 expression and soluble VAP-1 (sVAP-1) levels were assessed in the synovium synovial fluid and in the serum in cohorts of patients with tibiofemoral medial knee OA and healthy subjects. Standardized fixed-flexion poster anterior knee radiographs scored for Kellgren&ndash, Lawrence (KL) grade (0&ndash, 4) and medial joint space width (JSW). KL1/2 vs. KL3/4 scores defined early and advanced radiographic severity, respectively. Biochemical markers assessed in serum or synovial fluids (SF) comprised sVAP-1, interleukin 1 receptor antagonist (IL-1Ra), interleukin 6 (IL-6), soluble receptor for advanced glycation end-products (sRAGE), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 4 (CCL4), cluster of differentiation 163 (CD163), high sensitivity C-reactive protein (hsCRP), and matrix metalloproteinases (MMPs)-1,-3,-9. Associations between biomarkers and radiographic severity KL1/2 vs. KL3/4 (logistic regression controlling for covariates) and pain (Spearman correlation) were evaluated. Results: Elevated levels of sVAP-1 observed in OA synovial fluid and VAP-1 expression in synovium based on immunohistochemical, microarray, and real-time quantitative polymerase chain reaction (qRT-PCR) analyses. However, serum sVAP-1 levels in OA patients were lower than in controls and inversely correlated with pain and inflammation markers (hsCRP and soluble RAGE). Soluble VAP-1 levels in serum were also lower in radiographically advanced (KL3/4) compared with early KL1/2 knee SKOA patients. Conclusion: Local (synovial fluid) semicarbazide-sensitive amine oxidase (SSAO)/sVAP-1 levels were elevated in OA and correlated with radiographic severity. However, systemic (serum) sVAP-1 levels were lower in SKOA patients than normal and inversely correlated with pain and inflammation markers. Serum sVAP-1 levels were higher in early (KL1/2) compared with advanced (KL3/4) SKOA patients.

BRB powder and extracts have been shown to inhibit tumorigenesis in oral and other alimentary-tract sites in animal models; and have promising effects on markers of carcinogenesis in several human trials. The purpose of this study was to compare the inhibitory effects of BRB and two related compounds (protocatechuic acid and kaempferol) on mutagenesis and DNA adduct formation induced by the tobacco carcinogen dibenzo[a,l]pyrene (DBP) and its metabolites, DBP-diol and DBP-diol epoxide (DBPDE), in a rat oral epithelial cell line. Mutagenesis and DNA adduct formation induced by DBP, DBP-diol and DBPDE were monitored in rat oral epithelial and fibroblast cells, containing a lacI mutagenesis reporter gene; and the order of potency in both adduct formation and mutagenesis was DBPDE>>DBP-diol>>DBP in both cell types. In the fibroblasts for mutagenesis, the potencies (in units of mutants/10E5 pfu /uM) were: DBPDE, 16,320; DBP-diol, 25; DBP1.7, (above background); background was 2.9., Similar results were obtained for epithelial cells. Analysis of DNA adducts levels is still in progress and results thus far (in units of cis + trans isomers of anti-DBPDE-dA/10E6 A, are: DBPDE, 2096, DBP-diol ≈1.8, DBP < 1.0. The above results are consistent with mutagenesis being dependent on DNA adducts and indicate this cell culture model reflects initiation of carcinogenesis. The background level of mutagenesis was greater in the epithelial cells, so the fibroblasts were then used for subsequent mutagenesis experiments. As initiation by DBP and DBP-diol requires biotransformation to an ultimate mutagen/carcinogen (in this case DBPDE) we initially assayed inhibition of DBP-diol induced mutagenesis, so that effects of BRB on processes involved in activation, as well as effects on steps after formation of DBPDE would be detected. The parent compound, DBP, was not employed because it was very weakly mutagenic, and studies with DBPDE are still in progress. Treatment of cells with one µM DBP-diol led to an increase in mutant fraction from 2.9 +/- 0.8 to 25 +/- 2.3 mutants/10E5 pfu. When pretreated with BRB and its related compounds 4 hours before the addition of DBP-diol, significant inhibition of mutagenesis was observed. With 3 uM protocatechuic acid mutagenesis was reduced to 15.6 +/- 3.7 and with 3uM kaempferol to 13.3 +/- 4.1 mutants/10E5 pfu. Pretreatment of the cells with 25 ug/ml of an anthocyanin-enriched BRB extract (a concentration previously used in colon cancer cells), reduced mutagenesis to 9.8 +/- 0.35 mutants/10E5 pfu. These results suggest that BRB and specific related components will be protective against carcinogenesis induced by certain tobacco carcinogens. Supported by NIH grant #CA173465. Citation Format: Joseph B. Guttenplan, Wieslawa Kosinska, Tianzhen Han, Kun-Ming Chen, Shangmin Zhang, Krishnegowda Gowdahalli, Amin Shantu, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by the tobacco carcinogen, dibenzo(a,l)pyrene and its metabolites in cultured rat oral fibroblasts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2014-252

ULT cigarettes produce much lower levels of tars and particulate matter than FFLT cigarettes, but are generally not safer alternatives with respect to carcinogenesis, possibly because of compensatory smoking behavior. In a previous study (P.G. Sacks, et al., Food Chem Toxicol 49 (2011) 2348-2355) we reported that tobacco smoke particulate (TSP) extracts from FF, FFLT and ULT cigarettes exerted similar effects on the induction of phase I and II proteins and the activation of the tobacco carcinogen, benzo(a)pyrene (BaP) to genotoxic metabolites in primary oral epithelial cells when compared on a concentration basis, although ULT cigarettes were less effective on a per cigarette basis. Here we report that both a FFLT and an ULT TSP extract transform a human oral epithelial cell line to growth independence in soft agar, and enhance the transforming effects of BaP. At equal concentrations the ULT extract was more effective than the FFLT extract. A human oral leukoplakia cell line, MSKleuk1, was employed in these studies. Cells were treated twice over a two week period or 3 times over a 3 week period with: a) one uM BaP, b) 4ug/ml TSP extract, c) TSP extract + BaP, or d) vehicle control. One week after the last treatment cells were seeded on soft agar and were assayed either by counting colonies 3 weeks after seeding, or 8 days after seeding using an anchorage-independent growth assay (CytoSelect 96-Well Cell Transformation Assay Kit, Cell Biolabs). Results in initial experiments for the two week treatment were assayed by counting colonies, and the fraction of growth-independent cells for groups a-d resp. were: a, 5.5 +/- 0.2; b, 6.0 +/- 0.6; c, 6.6 +/- 0.3 and d, 0.78 +/- 0.3 (all in units of 10E-4). For the three week treatment the results were: a, 9.1 +/- 0.5; b, 9.2 +/- 1.2; c, 11.9, +/- 2.5; d, 1.4 +/- 0.7. The TSP extract was from a reference filtered cigarette, 2RF4. A further soft agar assay was carried out using the cell transformation assay; and TSP extracts from a commercial ULT and 2RF4 cigarettes were compared, in the presence and absence of BaP. The groups were a) 1 uM BaP, b) 4ug/ml 2RF4 extract, c) 4ug/ml 2RF4 extract + 1 uM BaP, d) 4ug/ml ULT extract, e) 4ug/ml ULT extract + 1uM BaP and f) vehicle control. All groups were treated 2x as described above. Results are expressed relative to the vehicle control which is set as 1. a, 1.9 +/- 0.4; b, 1.4 +/- 0.3; c, 2.2 +/- 0.4; d, 2.1 +/ 0.3; e, 2.6 +/- 0.6.The results of these show: 1) human oral epithelial cells can be transformed by BaP, and TSP extracts from FFLT and ULT cigarettes, 2) the effects of BaP and TSP extracts are approximately additive, 3) when compared on a concentration of particulate basis, ULT TSP extracts were more effective than FFLT TSP extracts. Unlike previous studies relating primarily to carcinogenesis initiation, ULT TSP extracts appear to more effective at transformation than FFLT TSP extracts. Citation Format: Tianzhen Han, Peter Sacks, Joseph B. Guttenplan. Comparative transforming effects of ultra low tar (ULT) and full flavor low tar (FFLT) cigarette smoke particulate extracts on human oral epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1582. doi:10.1158/1538-7445.AM2014-1582

In this study we investigated the effects of the combination sulforaphane (Sul) and resveratrol (Rev) on carcinogen metabolism to DNA adducts in rat mammary fibroblasts, and on DMBA-induced mutagenesis in mammary tissue from rats treated in vivo. The use of combinations of potential cancer chemopreventive agents has become one strategy to optimize chemoprevention. By targeting more than one pathway/process it may be possible to impact prevention by additive or synergistic effects, and utilize lower doses of agents that might have toxic effects at higher doses. We have previously tested a number of potential inhibitors of mutagenesis induced by the rat mammary carcinogen, 7,12-dimethylbenzanthrace (DMBA) in rat mammary epithelial and fibroblast cell lines (isolated by Dr. P.D. Josephy from lacI rats, Mutat Res, 497: 39-47, 2001). We had compared binary combinations of the agents, sulforaphane (Sul), resveratrol (Res), lipoic acid, epigallocatechin gallate, a vitamin C and E mix, and N-acetylcysteine. The agents alone had minor effects, but the combination of Sul and Res inhibited mutagenesis by about 40%. The current study extended this finding. In the metabolism study, HPLC of metabolites was employed; benzo(a)pyrene (BaP), rather than DMBA was used as the carcinogen, as standards of BaP metabolites, but not those of DMBA were available. BaP like DMBA is a carcinogenic polycyclic aromatic hydrocarbon. Fibroblasts were used, as these metabolized BaP much more effectively than epithelial cells. In the presence of 4uM BaP, in the absence of inhibitors, the cells generated 498 ± 83 pg/ml medium of the BaP-7,8 dihydrodiol -9,10 epoxide decomposition product, BaP 7,8,9,10 tetrol. This was reduced by 0.18uM Sul to 153 ± 48, by 4uM Res to 416 ± 87 and by a combination of the agents to 83 ± 37 pg/ml. The reduction was significant for Sul and for the combination. For the in vivo study rats were treated (6/group) by gavage with a single dose of 80 mg/kg DMBA. Two weeks before the DMBA treatment and continuing for 4 wks, rats received 50 mg/L Res, 120 mg/L Sul, and combinations of the agents at 50 and 120 mg/L or ½ the doses in drinking water. Eight wks after the DMBA treatment the rats were euthanized and mutagenesis in the cII gene was measured. The treatments and mutant fractions resp. were: DMBA alone, 9.1 ± 5.0; DMBA + Res, 8.9 ± 3.4; Sul, 8.2 ± 2.0; Res + Sul, 7.1 ± 2.8; Res + Sul, ½ dose, 7.9 ± 3.4; control, 1.3 ± 1.0. DMBA-induced mutagenesis was inhibited by both combination treatments, and Sul alone, although the inhibitions did not reach statistical significance, possibly because of large inter-animal variations. Taken together the results support the use of combinations of agents to enhance chemoprotection. Supported by the Susan Komen Foundation grant # KG080836. Citation Format: Peter G. Sacks, Wieslawa Kosinska, Mitaliben Contractor, Tian-Zhen Han, Joseph B. Guttenplan. Effects of a combination of resveratrol and sulforaphane on mutagenesis and DNA adduct formation in rat mammary tissue and a rat mammary fibroblast cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2013-4876