Lysyl Oxidase Gene Expression Research Articles

Pan-Cancer Analysis of the LOX Family Reveals that LOX Affects Tumor Prognosis by Affecting Immune Infiltration.

The lysyl oxidase (LOX) gene family encodes for a group of copper-dependent enzymes that play a crucial role in the cross-linking of collagen and elastin fibers in the extracellular matrix (ECM). Dysregulation of LOX gene expression has been implicated in various pathological conditions, including cancer. Several studies have shown that the LOX gene family is involved in cancer progression and metastasis. The goal of this article is to conduct a comprehensive analysis of the LOX family's role in pan-cancer multiplexes. We utilized pan-cancer multi-omics sequencing data from TCGA to investigate the relationship between LOX family genes and tumors at four different levels: mutation, copy number variation, methylation, and gene expression. In addition, we also examined the relationship between LOX family genes and tumors at the cell line level using tumor cell line sequencing data from CCLE. Taking into account the impact of LOX family genes on lung cancer, we developed a LOX family lung cancer prognostic model to forecast the disease's prognosis. Our findings revealed that LOXL2 had the highest mutation frequency in tumors, while all four LOX family genes experienced some degree of copy number variation in diverse tumors. We observed that LOX, LOXL1 to LOXL3 were predominantly highly expressed in tumors including LUAD. The expression trends of LOX and LOXL1 to LOXL3 were consistent across tumor cell lines, but differed somewhat from LOXL4. Utilizing 25 LOX family-related genes, we constructed a LOX family prognostic model that performed well in predicting the prognosis of lung cancer. Through pan-cancer analysis, we gain further knowledge of the role of LOX family genes in different tumors, offering a novel pathway for future research into the relationship between LOX family genes and tumors.

Estrogen-related receptor γ (ERRγ) is a key regulator of lysyl oxidase gene expression in mouse hepatocytes

Lysyl oxidase (LOX), the copper-dependent extracellular enzyme, plays a critical role in the regulation of protein cross-linking in the extracellular matrix (ECM). It is also involved in liver regeneration and liver fibrosis. However, the mechanism of LOX regulation in mouse hepatocytes is still unclear. Here, we identify a molecular mechanism showing that orphan nuclear receptor estrogen-related receptor γ (ERRγ) regulates LOX gene expression in the presence of the pro-inflammatory cytokine, interleukin 6 (IL6). IL6 significantly stimulated the expression of ERRγ and LOX in mouse hepatocytes. Overexpression of ERRγ increased LOX mRNA and protein levels. Moreover, knockdown of ERRγ attenuated IL6-mediated LOX gene expression at mRNA and protein levels. Overexpression of ERRγ or IL6 treatment upregulated LOX gene promoter activity, while knockdown of ERRγ decreased the IL6-induced LOX promoter activity. Furthermore, GSK5182, a specific ERRγ inverse agonist, inhibited the induction effect of IL6 on LOX promoter activity and gene expression in mouse hepatocytes. Overall, our study elucidates the mechanism involved in the LOX gene regulation by nuclear receptor ERRγ in response to IL6 in mouse hepatocytes, suggesting that, in conditions such as chronic inflammation, IL6 may contribute to liver fibrosis via inducing LOX gene expression. Thus, LOX gene regulation by the inverse agonist of ERRγ can be applied to improve liver fibrosis.

Expression of LOX Suggests Poor Prognosis in Gastric Cancer.

Background: Lysyl oxidase (LOX) is a key enzyme for the cross-linking of collagen and elastin in the extracellular matrix. This study evaluated the prognostic role of LOX in gastric cancer (GC) by analyzing the data of The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) dataset.Methods: The Wilcoxon rank-sum test was used to calculate the expression difference of LOX gene in gastric cancer and normal tissues. Western blot and immunohistochemical staining were used to evaluate the expression level of LOX protein in gastric cancer. Kaplan-Meier analysis was used to calculate the survival difference between the high expression group and the low expression group in gastric cancer. The relationship between statistical clinicopathological characteristics and LOX gene expression was analyzed by Wilcoxon or Kruskal-Wallis test and logistic regression. Univariate and multivariate Cox regression analysis was used to find independent risk factors affecting the prognosis of GC patients. Gene set enrichment analysis (GSEA) was used to screen the possible mechanisms of LOX and GC. The CIBERSORT calculation method was used to evaluate the distribution of tumor-infiltrating immune cell (TIC) abundance.Results: LOX is highly expressed in gastric cancer tissues and is significantly related to poor overall survival. Wilcoxon or Kruskal-Wallis test and Logistic regression analysis showed, LOX overexpression is significantly correlated with T-stage progression in gastric cancer. Multivariate Cox regression analysis on TCGA and GEO data found that LOX (all p < 0.05) is an independent factor for poor GC prognosis. GSEA showed that high LOX expression is related to ECM receptor interaction, cancer, Hedgehog, TGF-beta, JAK-STAT, MAPK, Wnt, and mTOR signaling pathways. The expression level of LOX affects the immune activity of the tumor microenvironment in gastric cancer.Conclusion: High expression of LOX is a potential molecular indicator for poor prognosis of gastric cancer.

Arterial stiffness and cardiac dysfunction in Hutchinson-Gilford Progeria Syndrome corrected by inhibition of lysyl oxidase.

Arterial stiffening and cardiac dysfunction are hallmarks of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), but the molecular regulators remain unknown. Here, we show that the LaminAG609G mouse model of HGPS recapitulates the premature arterial stiffening and early diastolic dysfunction seen in human HGPS. Lysyl oxidase (LOX) is up-regulated in the arteries of these mice, and treatment with the LOX inhibitor, β-aminopropionitrile, improves arterial mechanics and cardiac function. Genome-wide and mechanistic analysis revealed reduced expression of the LOX-regulator, miR-145, in HGPS arteries, and forced expression of miR-145 restores normal LOX gene expression in HGPS smooth muscle cells. LOX abundance is also increased in the carotid arteries of aged wild-type mice, but its spatial expression differs from HGPS and its up-regulation is independent of changes in miR-145 abundance. Our results show that miR-145 is selectively misregulated in HGPS and that the consequent up-regulation of LOX is causal for premature arterial stiffening and cardiac dysfunction.

Upregulation of miR-29a suppressed the migration and invasion of trophoblasts by directly targeting LOXL2 in preeclampsia.

Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and foetal morbidity and mortality, with a prevalence of 6-8% of pregnancies. Although the downregulation of lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2), which leads to reduced trophoblast cell migration and invasion through activation of the TGF-β1/Smad3/collagen pathway, is relevant to preeclampsia, the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas are not yet understood. This study aimed to investigate the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas. The expression of miRNAs, LOX and LOXL2 in preeclamptic placentas and control placentas was analysed by qPCR. Localisation of miR29a and LOXL2 in preeclamptic placentas was performed by RNA-Fluorescence in-situ hybridization assay. The direct regulation of LOXL2 by miR-29a was assessed by dual-luciferase reporter assays in human extravillous trophoblast cells (HTR8/SVneo). Cell migration and invasion were evaluated by Transwell assays in HTR8/SVneo cells. miR-29a expression was upregulated in preeclamptic placentas and negatively correlated with LOXL2 mRNA expression levels. RNA-Fluorescence in-situ hybridization assay revealed a clear overlap between miR-29a and LOXL2 in the placentas of preeclampic women. LOXL2 was a direct target gene of miR-29a, as confirmed by a dual-luciferase reporter assay in HTR8/SVneo trophoblast cells. miR-29a suppressed HTR8/SVneo trophoblast cell migration and invasion. LOXL2 overexpression reversed the inhibitory effects of miR-29a on HTR8/SVneo trophoblast cell migration and invasion. Our results suggest that the upregulation of miR-29a suppresses the migration and invasion of HTR8/SVneo trophoblast cells by directly targeting LOXL2 in preeclampsia.

Antifibrotic effects of sulforaphane treatment on gingival elasticity reduces orthodontic relapse after rotational tooth movement in beagle dogs.

ObjectiveIncreased gingival elasticity has been implicated as the cause of relapse following orthodontic rotational tooth movement and approaches to reduce relapse are limited. This study aimed to investigate the effects of sulforaphane (SFN), an inhibitor of osteoclastogenesis, on gene expression in gingival fibroblasts and relapse after rotational tooth movement in beagle dogs.MethodsThe lower lateral incisors of five beagle dogs were rotated. SFN or dimethylsulfoxide (DMSO) were injected into the supra-alveolar gingiva of the experimental and control group, respectively, and the effect of SFN on relapse tendency was evaluated. Changes in mRNA expression of extracellular matrix components associated with gingival elasticity in beagles were investigated by real-time polymerase chain reaction. Morphology and arrangement of collagen fibers were observed on Masson’s trichrome staining of buccal gingival tissues of experimental and control teeth.ResultsSFN reduced the amount and percentage of relapse of orthodontic rotation. It also decreased the gene expression of lysyl oxidase and increased the gene expression of matrix metalloproteinase (MMP) 1 and MMP 12, compared with DMSO control subjects. Histologically, collagen fiber bundles were arranged irregularly and were not well connected in the SFN-treated group, whereas the fibers extended in parallel and perpendicular directions toward the gingiva and alveolar bone in a more regular and well-ordered arrangement in the DMSO-treated group.ConclusionsOur findings demonstrated that SFN treatment may be a promising pharmacologic approach to prevent orthodontic rotational relapse caused by increased gingival elasticity of rotated teeth in beagle dogs.

Human umbilical cord-derived mesenchymal stem cells conditioned medium ameliorates CCl4-induced liver fibrosis through regulation of expression and activity of liver lysyl oxidase

The effects of human umbilical cord blood mesenchymal stem cells conditioned medium (hMSC-CM) on Lysyl oxidase (LOX) family gene expression and proteins were evaluated. Fifty-four male Wistar rats were divided into nine groups. The control groups received olive oil and phosphate-buffered saline (PBS) while untreated groups received CCl4. The treated groups received hMSC-CM after the 4th week of CCl4 injection. LOX family expression and proteins were evaluated by RT-PCR and western blot. CCl4 enhanced significantly the expression of LOX genes and proteins. hMSC-CM significantly decreased the expression of the LOX genes and proteins. hMSC-CM decreased cross-linked collagen in the treated groups. hMSC-CM improved liver fibrosis through decreased LOX family gene expression.

Could the “Stiff Rim Sign” Be an Indicator of Lysyl Oxidase Activity in Breast Cancer?

Background: Shear wave elastography (SWE) is a non-invasive and easily applicable imaging modality, which can provide quantitative information of tissue stiffness. Peritumoral high SWE elasticity values (stiff rim sign) has been reported in many studies. Lysyl oxidase (LOX) enzyme is implicated in the formation of peri-tumoral stiffness. Objectives: The aim of the study was to investigate the correlation between SWE measures with LOX gene expression levels in breast cancer patients. Patients and Methods: Forty seven women were included in the study. The lesions evaluated by SWE and ultrasound guided tru-cut biopsies were performed from both of the central and peripheral parts. SWE values, LOX family gene expression levels, histopathological features of the lesions, as well as axillary and distant metastasis statuses were evaluated statistically. Results: Thirty of the patients had breast cancer (BC) (the patient group) and 17 of them had fibroadenoma (the control group). Lysyl oxidase like1 (LOXL1) expression level in BC samples (central parts) were found to be significantly higher than the control group (P = 0.022). Stiff rim sign was present in all BC lesions and none of the control group. The elastography values of the patient group were significantly higher than the control group statistically (P 0.05) both for patient and control groups. Conclusion: Although there were no significant correlations between LOX expressions and SWE parameters in our study, axillary and distant metastasis were found to be correlated with SWE features, which emphasized the prognostic potential of SWE.

Lysyl oxidase assists tumor‑initiating cells to enhance angiogenesis in hepatocellular carcinoma.

A highly tumorigenic and malignant sub‑population of HCC containing tumor‑initiating cells (TICs) are defined by high self‑renewal and sphere formation ability. Lysyl oxidase (LOX) regulates various factors involved in extracellular matrix (ECM) maintenance, migration and angiogenesis. Certain reports have demonstrated the role of LOX in ECM crosslinking, however, the cancer‑promoting effects of LOX in HCC remain unclear, and whether LOX has a role in the regulation of angiogenesis in HCC TICs has not been elucidated. In the current study, RNA sequencing using next‑generation sequencing technology and bioinformatics analyses revealed that LOX gene expression was significantly upregulated in cell spheres. Sphere cells may form tumors with more vascular enrichment compared with tumors produced from adherent cells, as observed in a mouse xenograft model. LOX expression is correlated with increased vascular endothelial growth factor (VEGF) and platelet‑derived growth factor, as demonstrated by analyses of The Cancer Genome Atlas and Gene Expression Omnibus databases. Conditioned media obtained from LOX‑overexpressing tumor cells stimulated angiogenesis via secreted VEGF and enhanced the tube formation capacity of endothelial cells. Furthermore, these functional behaviors were blocked by the LOX inhibitor β‑aminopropionitrile. These findings provide novel mechanistic insight into the pivotal role of LOX in the regulation of TICs in HCC. Combination of LOX inhibitor with sorafenib is a potentially advantageous strategy for HCC therapy.

Comparative Evaluation of role of Lysyl oxidase gene (LOXG473A) expression in pathogenesis and malignant transformation of Oral Submucous Fibrosis.

BackgroundIdentification and comparison of gene expression of Lysyl oxidase (LOX) in oral submucous fibrosis and controls and to determine its role in Pathogenesis of Oral submucous fibrosis.Material and MethodsOf total sample size (n=127), the whole blood sample were collected from case and control group in citrate vial. It is centrifused and stored at -800C. We collected and isolated RNA from blood of case group (n=127) and age and sex matched control group (n=127) recruited on the basis of inclusion criteria. The cDNA was prepared for 127 samples which were processed for gene expression of Lysyl oxidase (LOX) in relation to housekeeping genes (Beta actin and 18srRNA) and its role in pathogenesis of Oral submucous fibrosis.ResultsIn relative expression (Normalized ratio),relatively 11 cases shown down-regulation of lysyl oxidase gene while 27 cases shows up-regulation of lysyl oxidase gene while in 89 cases there were no regulation i.e expression of lysyl oxidase gene in case group was of same degree of control. In non-relative expression results (Non-normalized ratio), the 38 cases shown down regulation of LOX gene while in 53 cases, it was up-regulated however in remaining 36 cases there was neither up-regulation nor down-regulation of Lysyl oxidase gene i.e the expression of LOX gene is null.ConclusionsIn oral submucous fibrosis, the expression of Lysyl oxidase gene is mixed type i.e either it will down regulate/upregulate or there will be no expression at all comparatively. However in majority of cases the upregulation of lysyl oxidase is relatively more common than down-regulation or non expression of Lysyl oxidase gene. Key words:Oral submucous fibrosis, lysyl oxidase, betel nut, premalignant disorders.

Expression profiling and microRNA regulation of the LKB1 pathway in young and aged lung adenocarcinoma patients.

Lung cancer in young patients appears to have distinct clinicopathological features. The present study focused on the role of the serine/threonine kinase liver kinase B1 (LKB1), a known tumor suppressor gene, and its miRNA regulation in lung adenocarcinoma, particularly in young versus elderly patients. A total of 88 patients with lung adenocarcinoma were retrospectively analysed. A simultaneous quantification was performed of the expression of LKB1 mRNA and 15 microRNAs (miRNA/miRs; miRs −93, −96, −34a, −34c, −214, −33a, −30b, −145, −182, −30c, −183, −29b, −29c, −153 and −138) involved in the LKB1 pathway, as well as of 5 identified target mRNAs [cyclin D1 (CCND1), catenin β-1 (CTNNB1), lysyl oxidase (LOX), yes-associated protein 1 (YAP1) and survivin], using NanoString technology. KRAS mutations were investigated by pyrosequencing analysis. Patients ≤50 years were defined as a younger group, while patients >50 years old as an older group (n=44/group). No difference between the two groups was identified in terms of survival times analysed using the Kaplan-Meier method or KRAS mutations. Subsequently, the LKB1 signalling pathway was focused on, as a target for therapy in lung adenocarcinoma, and assessed with regards to clinicopathological features; we found that LOX levels in adenocarcinoma patients were significantly associated with histological subtype (P=0.03), stage (P<0.0001) and prognosis (P=0.02 for disease-free interval and P=0.005 for overall survival), but not with age. Furthermore, the miRNA target prediction model indicated that miR-93 and miR-30b appeared to have functional binding sites and downregulate the gene expression of LKB1 and LOX, respectively. In conclusion, young patients appeared have similar survival rates to elderly patients. The assessment of LKB1, its downstream genes and its regulation by miRNAs may have an impact on future research on lung adenocarcinoma in young and elderly patients. Further investigations will be necessary to elucidate the potential of this pathway as a novel target for therapy.

Over-expression of lysyl oxidase is associated with poor prognosis and response to therapy of patients with lower grade gliomas

Lower grade gliomas (LGGs) have highly diverse clinical phenotypes. The histological grade and type are insufficient to accurately predict the clinical outcomes of patients with LGGs. Therefore, identification of biomarkers that can facilitate the prediction of clinical outcomes in LGGs is urgently needed. Gene expression of LOX has been identified as a biomarker for various cancers. However, the clinical significance of LOX expression in LGGs has not been investigated. In this study, we analyzed the glioma RNA-seq dataset from TCGA (The Cancer Genome atlas) and identified lysyl oxidase (LOX) as a potential biomarker for LGGs. Kaplan-Meier survival analysis revealed that high LOX expression is associated with worse overall survival and recurrence free survival in LGG patients. Besides, high LOX expression is associated with poor response to primary therapy, follow-up treatment, targeted molecular therapy, and radiation therapy. Univariate and multivariate Cox regression analyses further confirmed LOX expression as an independent prognostic factor for LGG patients. Finally, we observed that LOX expression is significantly correlated with EMT (epithelial to mesenchymal transition) and IDH1 status in LGGs. In conclusion, our analyses suggest that LOX expression is a potential biomarker for prognosis and therapeutic response in LGGs.

Crosslinking Enzyme Lysyl Oxidase Modulates Scleral Remodeling in Form-Deprivation Myopia

ABSTRACTPurpose: Scleral remodeling causes the excessive ocular elongation that underlies myopia. Lysyl oxidase (LOX), a copper-containing amine oxidase, can catalyze collagen and elastin crosslinking. The purpose of this study was to investigate the role of LOX in scleral remodeling in form-deprivation myopia (FDM).Methods: Seventy-five guinea pigs were randomly divided into five groups as follows: a normal control group, an FDM group, an FDM plus β-aminopropionitrile (BAPN) group, an FDM plus TGF-β1 (TGF-β1) group, and an FDM plus vehicle group. A translucent diffuser was used to induce FDM, and intravitreal injection was used to administer BAPN, TGF-β1 or vehicle. The scleral LOX and collagen gene and protein levels and the posterior scleral ultrastructure and biomechanics were measured.Results: In the FDM group, both the scleral LOX and collagen gene and protein levels were significantly lower than those in the control eyes. The collagen fibril diameters were significantly decreased in the FDM group compared with the diameters in the control group. A significant decrease in LOX gene and protein expression was observed after BAPN injection, and an increase was observed after TGF-β1 treatment compared with the levels in the FDM group. Additionally, the scleral collagen fibrils were significantly decreased in the BAPN-treated eyes but increased in the TGF-β1-treated eyes compared with the FDM eyes. The ultimate stress and Young’s modulus of the sclera were lowest in the BAPN group, followed by the FDM group and the TGF-β1 group. The ultimate strain (%) of the sclera was lowest in the TGF-β1 group, followed by the FDM group and the BAPN group.Conclusion: LOX expression was significantly lowered in myopic sclera. Modulating LOX expression induced a change in both the scleral collagen fibril diameter and the scleral biomechanics. Therefore, LOX may play a key role in the myopia scleral remodeling procedure.

Hypoxia triggers angiogenesis by increasing expression of LOX genes in 3-D culture of ASCs and ECs

ObjectivesThis study aimed to investigate the expression changes of LOX (lysyl oxidase) family genes, VEGFA, and VEGFB under hypoxic conditions in a co-culture system of ASCs (adipose-derived stromal cells) and ECs (endothelial cells). Materials and methodsASCs and ECs were co-cultured under hypoxic and normal oxygen conditions in a 1:1 ratio, and the formation of vessel-like was detected at 7 days. The transwell co-culture system was used and cell lysates were collected at 7 days after co-culture in hypoxic and normal oxygen condition. Semi-quantitative PCR was performed to quantify the mRNA expression of VEGFA, VEGFB, and the LOX genes (LOX, LOXL-1, LOXL-2, LOXL-3, and LOXL-4). Expression changes were determined by densitomery. ResultsEnhanced angiogenesis was detected in the co-culture of ASCs and ECs under hypoxic conditions. Among the genes screened, VEGFA, VEGFB, LOXL-1, and LOXL-3 in ECs, both mono-cultured and co-cultured, were significantly enhanced after culturing under hypoxic conditions. In ASCs, VEGFB, LOXL-1, and LOXL-3 were upregulated. ConclusionsContact co-culture between ASCs and ECs promotes angiogenesis under hypoxia. LOXL-1 and LOXL-3 expressions were increased in both ASCs and ECs and might play important roles in the enhanced angiogenesis promoted by hypoxia.

Abstract 764: LOX and LOXL2 inhibition as a treatment for ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women. Despite advances in our understanding of the molecular biology of EOC, patient survival has not significantly improved in decades and new treatments are needed. There is increased interest in therapeutic targeting of the tumor microenvironment (TME). The lysyl oxidase (LOX) family of enzymes are involved in extracellular matrix remodelling through crosslinking of collagen and elastin. They have been shown to play an important role in the TME of other cancers and are regulated by hypoxia, however little is known about their role in EOC. Therefore we aimed to investigate the role of LOX and LOXL2 in EOC using in vitro models. Tumor epithelial (n = 2) and matched cancer-associated fibroblast cell lines (n = 2) derived from an SV40-induced mouse model of EOC were generated. MTT assays were performed on all cell lines to assess the effect of LOX and LOXL2 inhibition on cell viability. The effect of LOX and LOXL2 inhibition on cancer cell migration and invasion towards cancer-associated fibroblast conditioned media was also investigated using transwell assays. RNA and protein were isolated and analysed from cells cultured in normoxic and hypoxic conditions to assess whether LOX and LOXL2 gene and protein expression is regulated by hypoxia. Inhibition of LOX and LOXL2 did not affect cell viability. LOX and LOXL2 inhibition significantly reduced the migration and invasion of EOC cells towards cancer-associated fibroblast conditioned media in vitro (p&amp;lt;0.05). Gene expression analysis comparing Lox and Loxl2 expression in all cell lines demonstrated a significant increase in Lox and Loxl2 expression in cells cultured in hypoxia (p&amp;lt;0.05). Similarly, cellular and secreted LOX and LOXL2 protein expression was increased in cells cultured in hypoxic conditions, supporting hypoxia as a regulator of LOX and LOXL2 expression. This study demonstrates a role for LOX and LOXL2 in promoting migration and invasion of EOC cells in vitro. The upregulation of LOX and LOXL2 in hypoxia suggests a role in facilitating tumor growth and progression within hypoxic tumors. These results provide support for the utility of LOX and LOXL2 inhibition as promising and novel therapeutic strategies in EOC. Citation Format: Angela Cho, Amanda L. Hudson, Samuel Yuen, Nham Tran, Viive M. Howell, Emily K. Colvin. LOX and LOXL2 inhibition as a treatment for ovarian cancer. [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 764.

Lysyl oxidase expression in cardiac fibroblasts is regulated by α2β1 integrin interactions with the cellular microenvironment

Lysyl oxidase (LOX) catalyzes crosslink formation between fibrillar collagens and elastins and an increase in LOX activity has been associated with cardiac fibrosis following myocardial infarction (MI). It has been previously reported that LOX expression is regulated by growth factors and cytokines including transforming growth factor (TGF-β1); however, it is unclear how the biophysical and biochemical properties of the cellular microenvironment affect LOX expression. In this study, we isolated rat cardiac fibroblasts (CF) and infarct cardiac fibroblasts (ICF), from healthy and 1-week post-MI left ventricular tissue respectively, and cultured them under varied substrate conditions in vitro to assess their influence on LOX expression. Culture of ICF on collagen I-coated plates increased LOX expression versus uncoated plates with an additional increase observed with the presence of TGF-β1. To further investigate the effect of integrin interactions with collagen I on LOX expression, we inhibited the α2β1 integrin from binding to collagen I and found gene and protein expression of LOX to be downregulated. Together, this demonstrates that the interaction of α2β1 integrin to collagen I in the cellular microenvironment can regulate expression of LOX. Further studies investigating additional integrin interactions may identify therapeutic targets for treating cardiac fibrosis.

Associations Between Elastography Findings and Clinicopathological Factors in Breast Cancer.

This study aimed to explore the clinical significance of breast tumor tissue stiffness based on ultrasound elastographic evaluation in clinical breast cancer. Tumor tissue stiffness is mainly regulated by interactions among tumor cells, stromal cells, and extracellular matrix and was recently regarded as a representative feature of tumor microenvironment. Basic research has already revealed that the tumor stiffness can lead to tumor progression; however, little is known about its clinical significance because thus far, no useful modality is available in the clinical setting. We investigated the tumor stiffness by strain elastography in 503 consecutive patients with invasive breast cancer. Correlations between stiffness and clinicopathological factors, including tumor size, lymph node involvement, tumor subtypes, and stromal-related genes' expressions in primary breast tumor, were statistically examined. We identified that clinical tumor stiffness significantly correlated with lymph node involvement and invasive tumor size but not with hormonal receptor expressions, human epidermal growth factor receptor type 2 status, and ki67 labeling index by analyses of both categorical and continuous variables of stiffness. On multivariate analyses, axillary lymph node metastasis was an independent factor that influenced the stiffness of primary breast tumor. In the gene expression analyses, relatively hard tumors had a significantly high gene expression of lysyl oxidase compared with soft tumors. Our study showed a close relationship between primary tumor stiffness by elastographic evaluation and lymph node involvement in clinical breast cancer. Further investigations on tumor-related tissue stiffness are required.

Mechanical Loading Stimulates Expression of Collagen Cross-Linking Associated Enzymes in Periodontal Ligament.

Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post-translationally modified by a series of specific enzymes. Among the collagen-modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross-linking and lysyl hydroxylases (LHs) to regulate the cross-linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen-modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intra-peritoneal injection of a LOX inhibitor, β-aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL-derived cells. The mechanical loading also elevated formation of collagen α-chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2-positive cells was observed only at the bone-side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up-regulating collagen-modifying enzymes and subsequent collagen cross-linking which are important for PDL tissue maintenance. J. Cell. Physiol. 231: 926-933, 2016. © 2015 Wiley Periodicals, Inc.

Abstract LB-009: Collagen regulation in postpartum mammary gland involution, a novel breast cancer prevention target

Abstract Collagen is the most abundant extracellular protein in breast tissue and plays a critical role in breast cancer progression. Intravital imaging shows collagen fibers as “highways” for tumor cell invasion, and collagen fibers arranged perpendicular to the tumor border (radial alignment) independently predict breast cancer metastasis. To understand the role of collagen in breast cancer, we argue it is necessary to understand collagen biology in the normal breast. While collagen production has been well studied in the context of wound healing, little is known about its regulation in normal breast physiology. Here we utilize a murine, weaning-induced mammary gland involution model, which is characterized by robust collagen deposition and remodeling, to investigate collagen regulation in the normal mammary gland. During mammary gland involution, we find increased collagen-I, -III, and -V gene expression and high levels of lysyl-oxidase (LOX), a major collagen cross-linker. By SHG analysis, we find collagen fibers around mammary ducts in the involuting gland are more tightly organized than nulliparous gland, with “hot spots” of radial alignment. Collagen is predominantly produced by fibroblasts and we identified mammary fibroblasts as PDGFRα+. Using flow cytometry-based cell sorting and gene expression analysis, PDGFRα+ fibroblasts isolated from the involuting mammary gland have increased gene expression of collagen-I,-III, and LOX. Further, there is ∼2 fold increase in number of PDGFRα+ cells/mg weight of tissue in the involuting gland, suggesting that fibroblasts may be recruited and activated during involution. Increased production of collagen by fibroblasts and radial alignment of collagen are consistent with a tumor promotional microenvironment during mammary gland involution. In fact, our lab has demonstrated pro-tumorigenic attributes of involution in rodent models of postpartum breast cancer, which can be mitigated by non-steroidal anti-inflammatory drug (NSAID) treatment. To investigate the impact of NSAID treatment on collagen during involution, we isolated fibroblasts from mammary glands of animals undergoing involution in the presence and absence of NSAID intervention. Mammary fibroblasts flow sorted from NSAID treated hosts display reduced collagen I, TGFβ1, and MMP-3 gene expression by 30∼60% compared to involution-fibroblasts isolated from untreated mice. Taken together, these data demonstrate that fibroblasts are responsive to COX-2 inhibition and suggest a novel mechanism of NSAIDs action that may be mediated through suppression of collagen deposition. Collagen and breast cancer are inseparable topics, as collagen abundance, architecture and tension impact breast cancer risk and prognosis. Weaning induced mammary gland involution provides a unique physiological window to study collagen regulation. Studying the mechanism of how NSAIDs regulate collagen may provide insight into the use of NSAIDs as a preventative and therapeutic intervention in breast cancer. Citation Format: Qiuchen Guo, Pepper J. Schedin. Collagen regulation in postpartum mammary gland involution, a novel breast cancer prevention target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-009. doi:10.1158/1538-7445.AM2015-LB-009

Abstract 367: Lysyl Oxidase Expression by Cardiac Fibroblasts is Regulated by Integrin-mediated Signaling

Cardiac fibrosis following myocardial infarction (MI) leads to reduced cardiac function, and contributes to heart failure and mortality. Recent studies shown the extent of adverse remodeling may be mitigated by therapeutic strategies which regulate cardiac fibroblast mediated-remodeling. Since cross-linking by lysyl oxidase (LOX) increases following MI and alters the mechanical properties of the infarct, it is critical to characterize how its expression is regulated by CFs post-MI. While LOX expression is attributable to TGF-β1 signaling, we hypothesize that changes in the stiffness and composition of the ECM can also alter LOX expression via integrin-mediated signaling. To investigate this, we isolated CFs from healthy left ventricle (LV) and infarcted cardiac fibroblasts (ICFs) from 1 week post-MI LV and cultured them on tissue culture plastic (TCP) and collagen I-coated plates (COL) in serum-free media for 48 hours to assess the expression of genes associated with LOX signaling, fibrosis, and myofibroblast activation. Our results show an upregulation of LOX gene expression in both CFs and ICFs when cultured on COL and this is further emphasized with the presence of TGF-β1 (Fig. 1A). Gene expression of col1α1, integrin β1 subunit and αSMA (Fig. 1B-D) also exhibit similar upregulation. Ongoing studies will investigate how altered substrate stiffness and composition affect gene expression of LOX and other genes associated with fibrosis. By understanding the effect of the physical microenvironment on the expression of fibrotic genes including LOX, we aim to develop novel therapeutic strategies to attenuate cardiac fibrosis and thus improve cardiac recovery following MI.