Mathematical modelling of blood-brain barrier failure and oedema.

Injuries such as traumatic brain injury and stroke can result in increased blood-brain barrier (BBB) permeability. This increase may lead to water accumulation in the brain tissue resulting in vasogenic oedema. Although the initial injury may be localized, the resulting oedema causes mechanical damage and compression of the vasculature beyond the original injury site. We employ a biphasic mixture model to investigate the consequences of BBB permeability changes within a region of brain tissue and the onset of vasogenic oedema. We find that such localized changes can indeed result in brain tissue swelling and suggest that the type of damage that results (stress damage or strain damage) depends on the ability of the brain to clear oedema fluid.

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase.

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.

Propagation of damage in brain tissue: coupling the mechanics of oedema and oxygen delivery.

Brain tissue swelling, or oedema, is a dangerous consequence of traumatic brain injury and stroke. In particular, a locally swollen region can cause the injury to propagate further through the brain: swelling causes mechanical compression of the vasculature in the surrounding tissue and so can cut off that tissue's oxygen supply. We use a triphasic mathematical model to investigate this propagation, and couple tissue mechanics with oxygen delivery. Starting from a fully coupled, finite elasticity, model, we show that simplifications can be made that allow us to express the volume of the propagating region of damage analytically in terms of key parameters. Our results show that performing a craniectomy, to alleviate pressure in the brain and allow the tissue to swell outwards, reduces the propagation of damage; this finding agrees with experimental observations.

Is the Donnan effect sufficient to explain swelling in brain tissue slices?

Brain tissue swelling is a dangerous consequence of traumatic injury and is associated with raised intracranial pressure and restricted blood flow. We consider the mechanical effects that drive swelling of brain tissue slices in an ionic solution bath, motivated by recent experimental results that showed that the volume change of tissue slices depends on the ionic concentration of the bathing solution. This result was attributed to the presence of large charged molecules that induce ion concentration gradients to ensure electroneutrality (the Donnan effect), leading to osmotic pressures and water accumulation. We use a mathematical triphasic model for soft tissue to characterize the underlying processes that could lead to the volume changes observed experimentally. We suggest that swelling is caused by an osmotic pressure increase driven by both non-permeating solutes released by necrotic cells, in addition to the Donnan effect. Both effects are necessary to explain the dependence of the tissue slice volume on the ionic bath concentration that was observed experimentally.

Tumor-secreted LOXL2 activates fibroblasts through FAK signaling.

UNLABELLED: Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular fibroblasts, it was determined that expression of α-SMA was localized to fibroblasts recruited from the host tissue. This marker also revealed that the matrix present in tumors with reduced levels of LOXL2 was more scattered compared with control tumors which exhibited matrices with dense, parallel alignments. Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α-SMA in fibroblasts grown on collagen matrices. Mechanistically, it was determined that LOXL2 activated fibroblasts through integrin-mediated focal adhesion kinase activation. These results indicate that inhibition of LOXL2 in tumors not only reduces tumor cell invasion but also attenuates the activation of host cells in the tumor microenvironment. IMPLICATIONS: These findings reveal new insight into the mechanisms of fibroblast activation, a novel function of LOXL2, and further highlight the importance of generating LOXL2-targeted therapies for the prevention of tumor progression and metastasis.

LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis.

Tumor metastasis is a highly complex, dynamic, and inefficient process involving multiple steps, yet it accounts for more than 90% of cancer-related deaths. Although it has long been known that fibrotic signals enhance tumor progression and metastasis, the underlying molecular mechanisms are still unclear. Identifying events involved in creating environments that promote metastatic colonization and growth are critical for the development of effective cancer therapies. Here, we show a critical role for lysyl oxidase (LOX) in establishing a milieu within fibrosing tissues that is favorable to growth of metastastic tumor cells. We show that LOX-dependent collagen crosslinking is involved in creating a growth-permissive fibrotic microenvironment capable of supporting metastatic growth by enhancing tumor cell persistence and survival. We show that therapeutic targeting of LOX abrogates not only the extent to which fibrosis manifests, but also prevents fibrosis-enhanced metastatic colonization. Finally, we show that the LOX-mediated collagen crosslinking directly increases tumor cell proliferation, enhancing metastatic colonization and growth manifesting in vivo as increased metastasis. This is the first time that crosslinking of collagen I has been shown to enhance metastatic growth. These findings provide an important link between ECM homeostasis, fibrosis, and cancer with important clinical implications for both the treatment of fibrotic disease and cancer.

Lysyl oxidase plays a critical role in endothelial cell stimulation to drive tumor angiogenesis.

Identification of key molecules that drive angiogenesis is critical for the development of new modalities for the prevention of solid tumor progression. Using multiple models of colorectal cancer, we show that activity of the extracellular matrix-modifying enzyme lysyl oxidase (LOX) is essential for stimulating endothelial cells in vitro and angiogenesis in vivo. We show that LOX activates Akt through platelet-derived growth factor receptor ß (PDGFRß) stimulation, resulting in increased VEGF expression. LOX-driven angiogenesis can be abrogated through targeting LOX directly or using inhibitors of PDGFRß, Akt, and VEGF signaling. Furthermore, we show that LOX is clinically correlated with VEGF expression and blood vessel formation in 515 colorectal cancer patient samples. Finally, we validate our findings in a breast cancer model, showing the universality of these observations. Taken together, our findings have broad clinical and therapeutic implications for a wide variety of solid tumor types.

The role of lysyl oxidase in SRC-dependent proliferation and metastasis of colorectal cancer.

BACKGROUND: Emerging evidence implicates lysyl oxidase (LOX), an extracellular matrix-modifying enzyme, in promoting metastasis of solid tumors. We investigated whether LOX plays an important role in the metastasis of colorectal cancer (CRC). METHODS: We analyzed LOX expression in a patient CRC tissue microarray consisting of normal colon mucosa (n = 49), primary (n = 510), and metastatic (n = 198) tissues. LOX was overexpressed in CRC cell line SW480 (SW480+LOX), and the expression was knocked down in CRC cell line SW620 using LOX-specific short hairpin RNA (SW620+shLOX). Effect of LOX manipulation on three-dimensional cell proliferation and invasion was characterized in vitro. Effect of LOX manipulation on tumor proliferation and metastasis was investigated in a subcutaneous tumor mouse model (n = 3 mice per group) and in an intrasplenic metastatic mouse model (n = 3 mice per group). The mechanism of LOX-mediated effects via v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) was investigated using dasatinib, an inhibitor of SRC activation. All statistical tests were two-sided. RESULTS: Compared with normal colon tissue (n = 49), LOX expression was statistically significantly increased in tumor tissues (n = 510) of CRC patients (P < .001), and a greater increase was observed in metastatic tissue (n = 198). SW480+LOX cells showed a statistically significantly increased three-dimensional proliferation (P = .037) and invasion (P = .015), whereas SW620+shLOX cells showed reduced proliferation (P = .011) and invasion (P = .013) compared with controls. Subcutaneous tumor growth in mice was statistically significantly increased in SW480+LOX tumors (P = .036) and decreased in SW620+shLOX tumors (P = .048), and metastasis was statistically significantly increased in SW480+LOX tumors (P = .044) and decreased in SW620+shLOX tumors (SW620 control vs SW620+shLOX, mean = 1.0 luminescent signal, 95% confidence interval = 0.3 to 1.7 luminescent signal, vs mean = 0.3 luminescent signal, 95% confidence interval = 0.1 to 0.5 luminescent signal; P = .035) compared with controls. LOX-mediated effects on tumor progression were associated with SRC activation, and these effects were inhibited by dasatinib. CONCLUSIONS: LOX showed an important role in CRC cell proliferation and metastasis and was dependent on the activation of SRC. These results have the potential to identify patients with high SRC activity, who may benefit from dasatinib treatment.

LOXL2-mediated matrix remodeling in metastasis and mammary gland involution.

More than 90% of cancer patient mortality is attributed to metastasis. In this study, we investigated a role for the lysyl oxidase-related enzyme lysyl oxidase-like 2 (LOXL2) in breast cancer metastasis, in both patient samples and in vivo models. Analysis of a published microarray data set revealed that LOXL2 expression is correlated with metastasis and decreased survival in patients with aggressive breast cancer. In immunocompetent or immunocompromised orthotopic and transgenic breast cancer models we showed that genetic, chemical or antibody-mediated inhibition of LOXL2 resulted in decreased metastasis. Mechanistic investigations revealed that LOXL2 promotes invasion by regulating the expression and activity of the extracellular proteins tissue inhibitor of metalloproteinase-1 (TIMP1) and matrix metalloproteinase-9 (MMP9). We found that LOXL2, TIMP1, and MMP9 are coexpressed during mammary gland involution, suggesting they function together in glandular remodeling after weaning. Finally, we found that LOXL2 is highly expressed in the basal/myoepithelial mammary cell lineage, like many other genes that are upregulated in basal-like breast cancers. Our findings highlight the importance of LOXL2 in breast cancer progression and support the development of anti-LOXL2 therapeutics for the treatment of metastatic breast cancer.

Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.

Tumor cell metastasis is facilitated by "premetastatic niches" formed in destination organs by invading bone marrow-derived cells (BMDCs). Lysyl oxidase (LOX) is critical for premetastatic niche formation. LOX secreted by hypoxic breast tumor cells accumulates at premetastatic sites, crosslinks collagen IV in the basement membrane, and is essential for CD11b+ myeloid cell recruitment. CD11b+ cells adhere to crosslinked collagen IV and produce matrix metalloproteinase-2, which cleaves collagen, enhancing the invasion and recruitment of BMDCs and metastasizing tumor cells. LOX inhibition prevents CD11b+ cell recruitment and metastatic growth. CD11b+ cells and LOX also colocalize in biopsies of human metastases. Our findings demonstrate a critical role for LOX in premetastatic niche formation and support targeting LOX for the treatment and prevention of metastatic disease.

c-Myb regulates lineage choice in developing thymocytes via its target gene Gata3.

During T-cell development, thymocytes with intermediate avidity for antigen-MHC complexes are positively selected and then differentiate into functional cytotoxic and helper T cells. This process is controlled by signalling from the T-cell receptor (TCR). Here, we show that the c-Myb transcription factor is a critical downstream regulator of positive selection, promoting the development of helper T cells and blocking the development of cytotoxic T cells. A gain-of-function c-Myb transgene stops development of cytotoxic T cells, instead causing accumulation of a precursor population. Conversely, loss of c-Myb in selecting cells results in significantly fewer helper T cells. In c-Myb-null thymocytes, Gata3, a critical inducer of T-helper cell fate, is not upregulated in response to T-cell receptor signaling, following selection. We show that Gata3 is a direct target of c-Myb, and propose that c-Myb is an important regulator of Gata3, required for transduction of the T-cell receptor signal for subsequent helper cell lineage differentiation.

Regulation of erythropoiesis by the neuronal transmembrane protein Lrfn2.

OBJECTIVE: The transgenic mouse line MEnTCD2.5 expresses a dominant interfering Myb protein in a T-cell-specific fashion. When MEnTCD2.5 animals are crossed to a second line ubiquitously expressing Myc, they develop a rapid onset, fatal disease characterized by enlarged lymph nodes full of nonlymphoid cells. This study aimed to elucidate the reason for this anomalous non-T-cell phenotype. MATERIALS AND METHODS: We studied the cells by morphological analysis, surface marker staining, mRNA expression studies and in vitro colony-forming assays. RESULTS: Aberrant cells in MEnTCD2.5 lymph nodes are erythroblasts, and cooperation between MEnTCD2.5 and Myc causes severe erythroblastosis, but not erythroleukemia. MEnTCD2.5:Myc and MEnTCD2.5 animals have pronounced extramedullary erythropoiesis in their lymph nodes, and some increase in bone marrow-derived erythroid progenitors; no other MEnTCD2 transgenic line cooperates in this fashion with Myc, suggesting that the MEnTCD2.5 integration site, in intron 2 of the Lrfn2 gene, is of importance. To confirm this, in in vitro colony-forming assays, expression of wild-type Lrfn2 phenocopies the MEnTCD2.5 defect. Finally, Lrfn2 expression also causes the outgrowth of a bizarre cell type in colony-forming assays that stains positively for both early hematopoietic and fibroblast/fibrocyte surface markers. CONCLUSIONS: The Lrfn2 protein, a transmembrane adhesion-type molecule, is able to subvert hematopoietic differentiation to increase erythropoiesis. In cooperation with Myc, this leads to erythroblastosis. Lrfn2 may also be involved in colony forming units-fibroblast regulation. As Lrfn2 expression is detectable in wild-type bone marrow, it likely plays a novel role during normal hematopoiesis.

Myb proteins regulate the expression of diverse target genes.

Hematopoiesis, the process by which mature blood cells arise, is controlled by multiple transcription factors, which act in stage- and lineage-specific complexes. It is a major goal to elucidate the genes regulated by these transcription factors, in order to obtain a full understanding of the process and its malignant counterpart, leukemia. Myb family transcription factors play a central role in hematopoiesis. To identify new Myb family target genes, we have used an inducible dominant-negative protein for a subtraction cloning protocol in a model cell system (FDCP-Mix) with many characteristics of normal hematopoiesis. We present here a novel group of 29 validated Myb family target genes of diverse functions.

A transcriptional regulatory element in the coding sequence of the human Bcl-2 gene.

We investigated the protein-binding sites in a DNAse I hypersensitive site associated with bcl-2 gene expression in human B cells. We mapped this hypersensitive site to the coding sequence of exon 2 of the bcl-2 gene in the bcl-2-expressing REH B-cell line. Electrophoretic mobility shift assays (EMSAs) with extracts from REH cells revealed three previously unrecognized B-Myb-binding sites in this sequence. The protein was identified as B-Myb by using a specific antibody and EMSAs. Accordingly, the levels of B-Myb and bcl-2 proteins, and of Myb EMSA activity, were correlated over a wide range of cell lines, representing different stages of B-cell development. Transfection of REH cells with antisense B-myb down-regulated EMSA activity and the level of bcl-2, and led to the apoptosis of REH cells. Transfection of the bcl-2-non-expressing RPMI 8226 cell line with a B-Myb expression vector induced B-Myb EMSA activity and the expression of bcl-2. Reporter assays indicated that the HSS8 sequence containing the three B-Myb sites may act as an enhancer when it is linked to the bcl-2 gene promoter. Interaction of B-Myb with HSS8 may enhance bcl-2 gene expression by co-operating with positive regulatory elements (e.g. previously identified B-Myb response elements) or silencing negative response elements in the bcl-2 gene promoter.