Reducible Collagen Cross-links Research Articles

Abstract 578: Novel bi-thiazole LOX inhibitors to overcome chemotherapy resistance in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype accounting for around 15% of all breast cancer patients and is responsible for 30% of breast cancer-related deaths. Although chemotherapy is the mainstay therapy for TNBC, development of resistance significantly reduces patients’ survival. Therefore, there is an urgent need to identify novel and effective treatment strategies to overcome chemoresistance. Recently, we identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX), a member of LOX family, as a key mediator of chemoresistance in TNBC. In this study, combining a robust screening platform for drug-like molecules and cell-based/recombinant protein assays, we identified bi-thiazole derivatives as novel potent LOX inhibitors. Further structure activity relationship (SAR) analysis resulted in two lead compounds: 6403, a relatively LOX-specific inhibitor, and 6415, a more LOX/LOXL2 dual inhibitor. Both compounds reduced collagen crosslinking and led to chemosensitization in TNBC cell lines in 3D culture and in chemoresistant TNBC PDX organoids. Furthermore, re-analyses of scRNA-Seq data of TNBC patients treated with neo-adjuvant chemotherapy with known chemoresponse or resistance showed that LOX+ cells were enriched upon chemotherapy only in treatment-refractory patients. Importantly, we identified a significant correlation between LOX and the gene sets of Reactive Oxygen Species (ROS) and DNA repair in TNBC patients treated with chemotherapy. Supporting this, increased chemotherapy penetration upon LOX inhibition resulted in elevated ROS levels and DNA damage in cells, leading to inhibition of FAK/Akt survival signaling. The efficacy of 6403 was tested in a chemoresistant TNBC PDX model where combination of doxorubicin with LOX inhibitor overcame doxorubicin resistance with no significant change in body weights. State-of-the-art MALDI-MSI and MP-SHG experiments showed efficient reduction of collagen content and crosslinking, respectively in PDX tumors upon LOX inhibition. Overall, these results show that novel bi-thiazole LOX inhibitors block collagen crosslinking and potentiates chemotherapy-induced ROS-DNA damage axis to overcome chemoresistance in TNBC. Citation Format: Metin Cetin, Ozge Saatci, Abdol-Hossein Rezaeian, Ozge Akbulut, Nageswara Rao Chintada, Harrison Taylor, Breanna Pederson, Ioulia Chatzistamou, Hamed Shateri Najafabadi, Susan Lessner, Peggi Angel, Campbell McInnes, Ozgur Sahin. Novel bi-thiazole LOX inhibitors to overcome chemotherapy resistance in triple negative breast 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 578.

Abstract P6-10-18: Developing novel lysyl oxidase (LOX) inhibitors to overcome chemotherapy resistance in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It accounts for ~15% of all breast cancer patients yet is responsible for 30% of breast cancer deaths. TNBC is treated primarily with conventional chemotherapy; however, resistance to therapy is common, leading to high mortality rates. Importantly, the benefit of current therapeutic strategies used in chemoresistant TNBC, i.e., immunotherapy and antibody-drug conjugates, is confined to only a fraction of patients, and survival benefit is limited. Therefore, there is an urgent need to identify novel and effective treatment strategies to overcome chemoresistance. Recently, we identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX), a member of LOX family, as a key mediator of chemoresistance in TNBC. However, currently available LOX inhibitors are either non-selective and/or show toxicity. Here, we performed a high-throughput cell-based LOX activity screen (HTS) with more than 5,000 molecules selected from a diversified compound library and identified the bi-thiazole derivatives as novel potent LOX inhibitors. Our structure activity relationship (SAR) analysis resulted in two lead compounds 6403, a relatively LOX-specific inhibitor, and 6415, a more LOX/LOXL2 inhibitor. Both compounds reduced collagen cross-linking (canonical function of LOX) and led to chemosensitization in TNBC cell lines and in chemoresistant TNBC PDX organoids. In addition, 6403 and 6415 reduced the TGF-beta induced fibrosis and inhibited migration capacity of the breast cancer cell lines. Importantly, 6403 showed excellent pharmacokinetic profile and did not lead to any observable toxicity in mice. Notably, 6403 overcame doxorubicin resistance in LOX-expressing 4T1 syngeneic model with no apparent toxicity. Furthermore, our novel LOX and LOX/LOXL2 dual inhibitors show superior inhibition of LOX activity compared to the recently developed and clinically tested LOX family or LOXL2/LOXL3 inhibitors. Overall, we identified novel potent LOX inhibitors with no observable toxicity for further preclinical development and future clinical testing to overcome chemoresistance in TNBC. Citation Format: Metin Cetin, Ozge Saatci, Ozge Akbulut, Chad Beneker, Abdol-Hossein Rezaeian, Mikhail Chernov, Campbell McInnes, Ozgur Sahin. Developing novel lysyl oxidase (LOX) inhibitors to overcome chemotherapy resistance in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-18.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

PSV-A-6 Poor Maternal Nutrition During Gestation Decreases Shear Force and Alters Gene Expression Related to Fibrogenesis

Abstract To test the hypothesis that poor maternal nutrition during gestation would alter offspring muscle shear force and adipogenesis, fibrogenesis, and myogenesis mRNA expression, Dorset ewes (n=46) pregnant with twins were fed 100%, 60% or 140% of NRC requirements from d30±0.02 of gestation until parturition. Male offspring from CON (n=12), OVER (n=13), and RES (n=21) ewes were euthanized at d284±1.82 and longissimus muscle (LM) was harvested for shear force and gene expression. Vacuum-sealed LM steaks were aged for 10 d postmortem (4℃) and tenderness was measured using Warner-Bratzler shear force (WBSF) methodology. RNA was isolated from the LM to quantify mRNA expression of genes related to adipogenesis (fatty acid binding protein 4, CCAAT/enhancer-binding protein α, and peroxisome proliferator-activated receptor γ), fibrogenesis [collagen A1A (COLA1A), COL3A1, cystain-c, fibronectin (FN1), and lysyl oxidase (LOX)], and myogenesis [bone-morphogenic protein (BMP)-1, myostatin, myogenin, and myogenic differentiation 1). The WBSF decreased by 21% and 18% in OVER relative to CON (P=0.01) and RES (P=0.03), respectively, but CON and RES were not different (P=0.60). Expression of BMP1 was 1.5- and 1.8-fold greater in RES than OVER and CON, respectively (P≤0.03). Expression of FN1 was 1.6-fold greater in RES than OVER (P=0.02). Expression of COL1A1 was 2.2-fold greater in RES than CON (P=0.05) and was 1.6-fold greater in RES than OVER (P=0.07). Expression of LOX was decreased 37% in OVER compared with CON (P=0.04) and decreased 21% compared with RES (P=0.08). An effect of diet was not observed for other genes (P≥0.18). In summary, RES offspring had greater expression of genes related to fibrogenesis, but no observed difference in tenderness relative to CON. Over-feeding during gestation increased meat tenderness, which may be due to reduced collagen cross-linking by LOX.

Abstract P504: Transient Angiotensin Converting Enzyme Inhibition Protects Against Future Fibrogenic Responses Via Sex-specific Mechanisms

Following washout of angiotensin converting enzyme inhibitor (ACEi) treatment in hypertensive rats (SHR), there is an increase in the proportion of homeostatic cardiac fibroblasts (CFs) characterized by a less fibrogenic gene profile. The present study investigated the impact of this shift in CFs on subsequent Ang II-induced oxidative stress and fibrogenic responses in the left ventricle (LV). Male and female SHRs (11 wk old) underwent a 6-week treatment scheme: 2-week ACEi (enalapril, 30mg/kg/day) or vehicle treatment, followed by a 2-week washout period, and then a 2-week Ang II (400ng/kg/min, s.c.) or vehicle infusion (n=5-11/group/sex). RT-qPCR for collagens and immunoblotting for LOX, Postn, OPN, NOX2, SOD2, and catalase was performed. In males, Ang II-induced increases in ColI, III, and IV expression were significantly attenuated by prior ACEi. While in females, Ang II significantly increased the expression of ColI and III similarly, regardless of prior ACEi treatment; highlighting a sex-specific impact of ACEi. After Ang II infusion, both Postn and LOX are reduced in LV of male and female SHR previously treated with an ACEi - suggesting reduced collagen cross-linking. Oxidative stress induced by NOX2 has been linked to OPN in fibrotic tissue. Ang II-induced increases in NOX2 are attenuated in LV of both sexes, while OPN is only reduced in females previously treated with an ACEi. NOX2 is significantly positively correlated with OPN in males only suggesting a sex-specific link between OPN and oxidative stress. Antioxidant (SOD2, catalase) levels were positively correlated to NOX2 in females, but not males. This suggests that females more efficiently neutralize oxidative stress than males - regardless of prior ACEi. Taken together, the persistent genomic and phenotypic changes in CFs following transient ACEi render the heart resistant to future fibrotic and oxidative effects of Ang II, but the mechanisms of protection may differ by sex. Collectively, these data reveal that there are sex-specific processes that govern collagen production vs. cross-linking and oxidative stress. Future studies will elucidate the mechanistic underpinnings of sex-specific cellular memory following transient ACEi with a goal of identifying novel therapeutic targets.

Glycosylated CD147 reduces myocardial collagen cross-linking in cardiac hypertrophy.

The mechanism of transition from chronic pressure overload-induced cardiac hypertrophy to heart failure is still unclear. Angiotensin II (Ang II) may be an important factor that mediates the transition in the end-stage of cardiac hypertrophy. In the present study, Goldblatt two-kidney one-clip (2K1C) rat model was used to simulate Ang II-induced hypertension. The elevated Ang II not only induced the concentric hypertrophy of left ventricle and cardiac fibrosis, but also increased the expression and glycosylation of CD147 in 2K1C rats. The left ventricular structure and function detected by echocardiogram showed a sign of the transition from cardiac hypertrophy to heart failure in 16 weeks of 2K1C rats. Ang II can activate N-acetylglucosamine transferase V (GnT-V), a key enzyme for CD147 glycosylation. Retinoic acid, an agonist of GnT-V, further increased glycosylated CD147, and activated matrix metalloproteinase-2/-9 (MMP-2 and MMP-9) in the hypertrophied left ventricle of 2K1C rat. Meanwhile, collagen cross-linking in the hypertrophied left ventricle significantly reduced in 2K1C rats. On the contrary, tunicamycin, an inhibitor of N-glycan biosynthesis, inhibited glycosylation of CD147 and activity of MMP-2 and MMP-9, and then maintained a stable of collagen cross-linking in the 2K1C rat hearts. The above results suggested that Ang II increased glycosylated CD147 which activated MMP-2 and MMP-9. Collagens were degraded by the activated MMPs and then reduced collagen cross-linking. Finally, the hypertrophied left ventricle was progressively dilated in chronic pressure overload due to losing the limitation of collagen cross-linking. Therefore, the compensated hypertrophy of left ventricle gradually transited to congestive heart failure.

Hepatitis transactivator protein X promotes extracellular matrix modification through HIF/LOX pathway in liver cancer

Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.

Abstract 4520: Hepatitis B virus X protein regulates hypoxia-inducible factor-1alpha (HIF-1 alpha) and lysyl oxidase like 2 (LOXL2) pathway in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a fatal malignancy and is prevalent in HBV-endemic geographical areas. Hepatitis B virus (HBV) infection is a major etiologic factor of HCC. HBV is a double-stranded DNA virus and encodes for a viral onco-protein called transactivator protein X (HBx) which interacts with the host proteins to enhance proliferative potential of the host cells. Here, using Tet-ON and Tet-OFF HBx inducible systems in HBV-negative HCC cell lines, we showed that induced expression of HBx stabilizes hypoxia-inducible factor-1α (HIF-1α) protein under hypoxia. Reversely, knockdown of HBx in HBV-positive HCC cell lines reduced HIF-1α protein stabilization under hypoxia. More intriguingly, we found that induced HBx expression up-regulated the mRNA and protein expression a known HIF-1α transcriptional target, lysyl oxidase like 2 (LOXL2) in HCC. Our previous study showed that LOXL2 cross-linked collagen in the extracellular matrix to promote HCC metastasis. Scanning electron microscopy demonstrated that knockdown of HBx in HCC cell lines markedly reduced formation of collagen fibers in vitro. Transwell invasion assay showed that knockdown of HBx reduced HCC invasive ability. Picrosirius red staining further showed that knockdown of HBx reduced collagen cross-linking in vivo and repressed HCC growth and metastasis. Taken together, our study unprecedentedly show the HBx remodels the ECM through HIF-1α/LOXL2 pathway to promote HCC metastasis. Citation Format: Cerise Yuen-Ki Chan, Aki Pui-Wah Tse, Elley Yong-Tuen Chiu, Karen Man-Fong Sze, Hui-Yu Koh, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Hepatitis B virus X protein regulates hypoxia-inducible factor-1alpha (HIF-1 alpha) and lysyl oxidase like 2 (LOXL2) pathway in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4520. doi:10.1158/1538-7445.AM2017-4520

Tissue transglutaminase induction in the pressure-overloaded myocardium regulates matrix remodelling.

Tissue transglutaminase (tTG) is induced in injured and remodelling tissues, and modulates cellular phenotype, while contributing to matrix cross-linking. Our study tested the hypothesis that tTG may be expressed in the pressure-overloaded myocardium, and may regulate cardiac function, myocardial fibrosis and chamber remodelling. In order to test the hypothesis, wild-type and tTG null mice were subjected to pressure overload induced through transverse aortic constriction. Moreover, we used isolated cardiac fibroblasts and macrophages to dissect the mechanisms of tTG-mediated actions. tTG expression was upregulated in the pressure-overloaded mouse heart and was localized in cardiomyocytes, interstitial cells, and in the extracellular matrix. In contrast, expression of transglutaminases 1, 3, 4, 5, 6, 7 and FXIII was not induced in the remodelling myocardium. In vitro, transforming growth factor (TGF)-β1 stimulated tTG synthesis in cardiac fibroblasts and in macrophages through distinct signalling pathways. tTG null mice had increased mortality and enhanced ventricular dilation following pressure overload, but were protected from diastolic dysfunction. tTG loss was associated with a hypercellular cardiac interstitium, reduced collagen cross-linking, and with accentuated matrix metalloproteinase (MMP)2 activity in the pressure-overloaded myocardium. In vitro, tTG did not modulate TGF-β-mediated responses in cardiac fibroblasts; however, tTG loss was associated with accentuated proliferative activity. Moreover, when bound to the matrix, recombinant tTG induced synthesis of tissue inhibitor of metalloproteinases (TIMP)-1 through transamidase-independent actions. Following pressure overload, endogenous tTG mediates matrix cross-linking, while protecting the remodelling myocardium from dilation by exerting matrix-preserving actions.

Selective targeting of lysyl oxidase-like 2 (LOXL2) suppresses hepatic fibrosis progression and accelerates its reversal

Background/AimsWe studied the role of lysyl oxidase-like 2 (LOXL2) in collagen crosslinking and hepatic progenitor cell (HPC) differentiation, and the therapeutic efficacy of a LOXL2-blocking monoclonal antibody on liver fibrosis...

Abstract LB-349: Copper depletion as a strategy to affect the tumor microenvironment in breast cancer patients at high risk of relapse and in triple negative preclinical models of breast cancer: Updated results of a phase II study of tetrathiomolybdate (TM) in breast cancer (BC) patients (pts) at high risk for recurrence

Abstract Background: The pre-metastatic niche mobilizes copper to assist in conditioning the tumor microenvironment facilitating tumor progression. This includes bone marrow derived VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent lysyl oxidase (LOX). We hypothesized that TM-associated CD would reduce EPCs and other copper dependent processes in the pre-metastatic niche in BC pts. Methods: Stage 2 triple negative BC, Stage 3 and stage 4 without any evidence of disease, (NED) BC pts, received oral TM to maintain ceruloplasmin (Cp) between 8-17mg/dL for 2 years or until relapse. Endpoints included change in EPCs, safety, disease-free (DFS) and overall survival (OS) and effect on biomarkers. Laboratory studies: MDA-LM2-luciferase cells were implanted into CB17-SCID mice, and treated with TM or water. Tumor progression was quantified by bioluminescence imaging (BLI), CD status by Cp oxidase levels, lysyl oxidase (LOXL2) activity by ELISA, and collagen deposition by Picrosirius Red staining and Second Harmonic Generation (SHG) imaging analysis. Results: 75 pts (45 stage 2/3 BC, 30 stage 4 NED) received over 2400 cycles of TM on the primary and extension studies. Median age: 51 yrs (range 29-66). Of the 75 pts, 48% had TNBC, 40% were stage 4 NED. Median Cp level decreased from 28 at baseline to 15.5 (p<0.0001) after one cycle. Grade 3/4 toxicities included reversible neutropenia (2.3%), anemia (0.04%), leukopenia (1.2%) and fatigue (0.09%). CD was associated with a decrease in EPCs (p = 0.0014) and serum LOX (p<0.001). At a median follow-up of 5.9 years, the PFS for all 75 pts is 72%, including a PFS of 90% for all stage 2/3 pts with TNBC. The overall survival is 84%. Relapse after two years is a rare event. In pre-clinical models, TM had no effects on the primary tumor, but decreased secondary lung metastases (p = 0.04) as seen by BLI. Western blots demonstrated decreased LOX in the pre-metastatic lungs vs controls (p = 0.03), and IHC showed reduced collagen cross-linking by image J (p = 0.01). SHG analysis showed reduced collagen fiber length. Conclusions: TM is safe, well tolerated and appears to affect multiple copper dependent biologic processes in the tumor microenvironment known to be important for tumor progression, most striking in TNBC. Randomized trials in a high risk for relapse population are warranted. Citation Format: Eleni Nackos, Sharrell Lee, Amy Willis, Naomi Kornhauser, Maureen Ward, Marta Cobham, Tessa Cigler, Anne Moore, Veronica Fitzpatrick, Sarah Schneider, Alysia Wiener, Jessica Guillaume-Abraham, Bo Ri Seo, J David Warren, Anna Rubinchik, Claudia Fischbach, Vivek Mittal, Linda Vahdat. Copper depletion as a strategy to affect the tumor microenvironment in breast cancer patients at high risk of relapse and in triple negative preclinical models of breast cancer: Updated results of a phase II study of tetrathiomolybdate (TM) in breast cancer (BC) patients (pts) at high risk for recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-349.

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.

Abstract 19973: Decellularized Extracellular Matrix Exhibits Reduced Stiffness and Immature Collagen Deposition in a Mouse Model of Left Heart Failure

The extracellular matrix (ECM) is a major determinant of myocardial mechanics in heart failure. Existing methods to evaluate ECM composition are limited. Our objective was to perform a non-destructive microstructural and mechanical analysis of de-cellularized ECM in a mouse model of left ventricular (LV) failure.Methods and Results: Wild-type mice were subjected to 8 weeks of trans-aortic constriction (TAC; n=6) and compared to sham operated controls (Sham; n=5). Compared to sham, TAC mice had increased LV and lung weights, increased LV end-diastolic pressure (4±2 vs 25±5 mmHg, p<0.01) and reduced dP/dT (8249±596 vs 4786±984 mmHg/sec, p<0.01). LV collagen abundance by picrosirius red staining was higher in TAC mice (1±0.1 vs 35±12%, p<0.05) and Type I collagen mRNA and protein levels were increased by 80-fold and 2-fold, respectively (p<0.05 for both). Using non-linear optical microscopy, collagen fiber organization, content and cross-linking were quantified from decellularized sections of the LV free wall. Using cumulative second harmonic generation (SHG), a trend to higher total collagen content per tissue volume was observed in TAC mice (0.02±0.01 vs 0.05±0.3, p=0.09). Two photon excited fluorescence within SHG positive voxels was used to measure cross-linking per collagen fiber. Compared to sham, reduced cross-linking was observed in TAC mice (0.2 ±0.1 vs. 0.11±0.04, p=0.02). Next, using tensile mechanical testing no difference in average ECM stiffness (elastic modulus) was observed between groups (31±15 vs 24±7, sham vs TAC, p=0.19). Conclusions: This is the first mechanical analysis of decellularized ECM in a murine model of LV failure. Despite increased Type I collagen mRNA, protein and histologic fibrosis after TAC, the ECM composition analysis showed reduced collagen cross-linking suggestive of functionally immature collagen deposition. Functional maturation of the ECM may be an important target of therapy for heart failure.

Abstract 2000: Lysyl oxidase-like 2 promotes hepatocellular carcinoma metastasis by remodelling the tumor and metastatic microenvironments

Abstract Hepatocellular carcinoma is a common and lethal malignancy worldwide. High death rate in HCC is mainly due to metastasis. This study revealed the detailed molecular mechanisms by which primary HCC promoted metastasis through formation of tumor and metastatic niches via lysyl oxidase-like 2 (LOXL2). LOXL2 is a secretory protein that belongs to the LOX family which is consisted of 5 members. LOX family members are amine oxidases that cross-link components in extracellular matrix (ECM) such as collagen and elastin. Among the five LOX family members, LOXL2 is the sole member over-expressed in HCC tissues. Remarkably, we detected a significant up-regulation of LOXL2 protein in the sera of 80 HCC patients as compared to normal individuals. Clinicopathological analysis showed that up-regulation of LOXL2 in human HCC was significantly associated with aggressive HCC feature, direct liver invasion. Interestingly, we found that LOXL2 expression in HCC cells was positively regulated by the hypoxia/HIF-1α and TFGβ/SMAD4-mediated pathways and negatively regulated by miR-26 and miR-29, two microRNAs families that are under-expressed in HCC. Abrogation of the LOXL2 pathway suppressed collagen cross-linking, bone marrow cell invasion, and HCC cell invasion in vitro. Stable knockdown of LOXL2 repressed tumor growth and intrahepatic metastasis in orthotopic tumor implantation model. Knockdown of LOXL2 also reduced collagen cross-linking and tumor stiffness in vivo. LOXL2-mediated change of tension within the tumor microenvironment activated a key cell motility regulator, rho-kinase (ROCK), and ROCK-mediated events such as actin polymerization and focal adhesion formation. In addition, we found that primary HCC tumor facilitated metastasis by directing bone marrow-derived cells (BMDCs) to colonize the distal metastatic sites prior to the arrival of HCC cells. We observed a profound reduction of collagen cross-linking, CD11b+ BMDC recruitment, and subsequent metastasis in the lungs of the mice that were orthotopically implanted with the LOXL2 knockdown HCC tumors. Conclusion: Our study highlighted three important advances in understanding the cancer biology of hepatocellular carcinoma. First, LOXL2 is overexpressed in HCC clinical samples and could be detected in sera of patients, indicating that LOXL2 may be a good diagnostic marker for HCC patients. Second, we delineated a complex interlinked signaling network that involves multiple regulators including HIF-1α/TGFβ/ microRNAs which converge to control the expression of LOXL2 leading to HCC progression. Third, we showed that LOXL2 played an imperative role in the tumor microenvironment and metastatic niches formation leading to HCC progression. We comprehensively unveiled the molecular mechanisms which drive LOXL2 dysregulation and the functional implications of LOXL2 in human HCC. Citation Format: Carmen Chak Lui Wong, Irene Oi-Lin Ng. Lysyl oxidase-like 2 promotes hepatocellular carcinoma metastasis by remodelling the tumor and metastatic microenvironments. [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 2000. doi:10.1158/1538-7445.AM2014-2000

Abstract CT309: Influencing the tumor microenvironment: A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence

Abstract Background: Bone marrow derived endothelial progenitor cells (EPCs) and copper-dependent angiogenic pathways are critical to the metastatic process. Copper depletion (CD) therapy inhibits tumor metastases in preclinical models. We hypothesized that TM-associated CD would reduce EPCs in pts at high risk for BC recurrence, and we explored the relationship between CD and its effects on the tumor microenvironment in pre-clinical models. Methods: In this single arm, phase II study, BC pts at high risk for recurrence, defined as node positive triple negative (TN), stage 3 and 4 with no evidence of disease (NED) were enrolled on a trial of CD with TM. We CD’d to maintain ceruloplasmin (Cp) between 5-17 mg/dl for 2 years or until relapse. The primary endpoint was change in EPCs measured before and during treatment with TM. Secondary endpoints included tolerability, safety, and efficacy of CD. Laboratory studies: MDA-LM2-luciferase cells were implanted into CB17-SCID mice gavaged with water or TM. The tumors were quantified by bioluminescence images (BLI). We measured Cp oxidase to determine copper status. Western blots were used to assess LOX activity, and IHC was used to quantify collagen cross-linking and CD11b+ macrophage infiltration. Results: We enrolled 43 pts. Treatment duration was 24 cycles (each cycle is 28 days) for the primary study. A total of 752 cycles were completed in 2 years. The mean age was 49 (range 29-66). Mean Cp level decreased from 29 at baseline to 16 (p<0.001) at 4 weeks. 94% of pts achieved CD by cycle 2. TM was well tolerated in most pts and the only grade 3 and 4 toxicities were neutropenia (3.9%) and anemia (0.1%). CD was most effective in TNBC pts with significantly reduced time to CD (P=0.0153). At one year of analysis, TM reduced EPCs (p=0.044). The 2-year analysis of effect of TM on EPCs is ongoing. The 2 year PFS for the entire cohort from the start of TM treatment was 81%. The overall survival from the start of treatment was 90.6% with median follow up of 4.5 years. In pre-clinical models, TM had no effects on the primary tumor, but decreased secondary metastases as seen by BLI. Western blots demonstrated decreased LOX, and IHC showed reduced collagen cross-linking with less CD11b+ macrophage recruitment.Conclusion: TM is safe, well tolerated and effective in achieving CD in breast cancer patients, and appears to affect the tumor microenvironment in pre-clinical models. Molecular subtype may affect CD and TM may be most effective in TN patients. Citation Format: Nancy Chan, Naomi Kornhauser, Maureen Ward, Amy Willis, Tessa Cigler, Ellen Chuang, Anne Moore, Diana Donovan, Sarah E. Schneider, Christina Lam, David J. Warren, Anna Rubinchik, Sandra Hurtado Rua, Sharrell Lee, Maureen Lane, Vivek Mittal, Linda Vahdat. Influencing the tumor microenvironment: A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence. [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 CT309. doi:10.1158/1538-7445.AM2014-CT309

Homocysteine induces serum amyloid A3 in osteoblastsviaunlocking RGD‐motifs in collagen

Hyperhomocysteinemia is a risk factor for osteoporotic fractures. Homocysteine (Hcys) inhibits collagen cross-linking and consequently decreases bone extracellular matrix (ECM) quality. Serum amyloid A (A-SAA), an acute-phase protein family, plays an important role in chronic and inflammatory diseases and up-regulates MMP13, which plays an important role in bone development and remodeling. Here, we investigate the effect of Hcys on expression of SAA3, a member of the A-SAA gene family, in osteoblasts characterizing underlying mechanisms and possible consequences on bone metabolism. MC3T3-E1 osteoblast-like cells were cultured up to 21 d with Hcys (low millimolar range) or reseeded onto ECM resulting from untreated or Hcys-treated MC3T3-E1 cells. Fourier-transformed infrared spectroscopy and a discriminative antibody were used to characterize the resulting ECM. Gene expression and signaling pathways were analyzed by gene chip, quantitative RT-PCR, and immunoblotting. Transcriptional regulation of Saa3 was studied by promoter transfection assays, chromatin immunoprecipitation, and immunofluorescence microscopy. Hcys treatment resulted in reduced collagen cross-linking, uncovering of RGD-motifs, and activation of the PTK2-PXN-CTNNB1 pathway followed by RELA activation. These signaling events led to increased SAA3 expression followed by the production of MMP13 and several chemokines, including Ccl5, Ccl2, Cxcl10, and Il6. Our data suggest Saa3 as link between hyperhomocysteinemia and development of osteoporosis.

Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation

Primary tumors facilitate metastasis by directing bone marrow-derived cells (BMDCs) to colonize the lungs before the arrival of cancer cells. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of breast cancer metastatic niche formation through induction of multiple members of the lysyl oxidase (LOX) family, including LOX, LOX-like 2, and LOX-like 4, which catalyze collagen cross-linking in the lungs before BMDC recruitment. Only a subset of LOX family members was expressed in any individual breast cancer, but HIF-1 was required for expression in each case. Knockdown of HIF-1 or hypoxia-induced LOX family members reduced collagen cross-linking, CD11b(+) BMDC recruitment, and metastasis formation in the lungs of mice after orthotopic transplantation of human breast cancer cells. Metastatic niche formation is an HIF-1-dependent event during breast cancer progression.

Impact of collagen crosslinking on the second harmonic generation signal and the fluorescence lifetime of collagen autofluorescence

Collagen is the major structural protein of the skin and its crosslinks are essential for its mechanical stability. In photodamaged skin, a decrease of the mature collagen crosslink histidinohydroxylysino-norleucine was reported. In this study, we investigated the consequences and measurability of the reduced crosslinking. In order to determine the consequences of reduced collagen crosslinking, in vitro models of reduced collagen crosslinking were established. The collagen synthesis and structure was analyzed using the signals second harmonic generation (SHG) and the fluorescence lifetime of the collagen autofluorescence by a multiphoton laser scanning microscope. Reduced collagen crosslinking results in a posttranscriptionally diminished collagen synthesis, a modified structure of the collagen fibers and fibrils and a higher intensity of the SHG signal. The SHG signal might be influenced by the interspaces of the collagen molecules within one collagen fibril. Because of these findings, it can be speculated that reduced collagen crosslinking changes the interspace of single collagen molecules within the collagen fibril, resulting in an enhanced SHG signal. Alternative explanations are discussed. Furthermore, the fluorescence lifetime was reduced in the in vitro models of reduced collagen crosslinking. In the crosslink sites of the collagen molecules, the main ratio of fluorescence is found. As the fluorescence lifetime is determined not only by the fluorescent molecule itself but also by its microenvironment, the change in the fluorescence lifetime might be explained by reduced crosslinking at the crosslink site. A reduction of collagen crosslinking (as seen in photodamaged skin) results in an increase of the SHG signal and a decrease of the fluorescence lifetime in vitro. In vivo measurements of the two parameters might reveal the status of collagen crosslinking and therefore help to identify the status of dermal photodamage or pathogenesis using collagen crosslinking determination.

Interaction between Periostin and BMP-1 Promotes Proteolytic Activation of Lysyl Oxidase

Intra- and intermolecular covalent cross-linking between collagen fibrils, catalyzed by lysyl oxidase (LOX), determines the mechanical properties of connective tissues; however, mechanisms that regulate the collagen cross-linking according to tissue specificity are not well understood. Here we show that periostin, a secretory protein in the dense connective tissues, promotes the activation of LOX. Previous studies showed that periostin null mice exhibit reduced collagen cross-linking in their femurs, periosteum, infarcted myocardium, and tendons. Presently, we showed that active LOX protein, formed by cleavage of its propeptide by bone morphogenetic protein-1 (BMP-1), was decreased in calvarial osteoblast cells derived from periostin null mice. Overexpression of periostin promoted the proteolytic cleavage of the propeptide, which increased the amount of active LOX protein. The results of co-immunoprecipitation and solid phase binding assays revealed that periostin interacted with BMP-1. Furthermore, this interaction probably resulted in enhanced deposition of BMP-1 on the extracellular matrix, suggesting that this enhanced deposition would lead to cleavage of the propeptide of LOX. Thus, we demonstrated that periostin supported BMP-1-mediated proteolytic activation of LOX on the extracellular matrix, which promoted collagen cross-linking.

PDGF‐BB released in tendon repair using a novel delivery system promotes cell proliferation and collagen remodeling

The purpose of this study was to promote fibroblast proliferation and collagen remodeling in flexor tendon repair through sustained delivery of platelet derived growth factor (PDGF-BB). The release kinetics of PDGF-BB from a novel fibrin matrix delivery system was initially evaluated in vitro. After the in vivo degradation rate of the fibrin matrix was determined using fluorescently tagged fibrin, PDGF-BB was delivered to the site of flexor tendon repair in vivo in a canine model. The effect of PDGF-BB on intrasynovial tendon healing was studied using histology-based assays (cell density, proliferation, and type I collagen expression) and by measuring total DNA levels and reducible collagen crosslink levels. The fibrin matrix delivery system provided sustained release of PDGF-BB in vitro at a rate modulated by the ratio of heparin to growth factor. In vivo, the fibrin matrix remained at the repair site for more than 10 days. Delivery of PDGF-BB led to a qualitative increase in cell density, cell proliferation, and type I collagen mRNA expression. PDGF-BB also led to statistically significant increases in total DNA (20% increase at 7 days, 18% increase at 14 days) and reducible collagen crosslinks (30% increase at 7 days). Sustained delivery of growth factors may be achieved using a novel fibrin-based delivery system. PDGF-BB delivery increased cell proliferation and matrix remodeling and thus may accelerate flexor tendon healing.