The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC.

Muscle is an integrated tissue composed of distinct cell types and extracellular matrix. While much emphasis has been placed on the factors required for the specification of the cells that comprise muscle, little is known about the crosstalk between them that enables the development of a patterned and functional tissue. We find in mice that deletion of lysyl oxidase (Lox), an extracellular enzyme regulating collagen maturation and organization, uncouples the balance between the amount of myofibers and that of muscle connective tissue (MCT). We show that Lox secreted from the myofibers attenuates TGFβ signaling, an inhibitor of myofiber differentiation and promoter of MCT development. We further demonstrate that a TGFβ-Lox feedback loop between the MCT and myofibers maintains the dynamic developmental homeostasis between muscle components while also regulating MCT organization. Our results allow a better understanding of diseases such as Duchenne muscular dystrophy, in which LOX and TGFβ signaling have been implicated and the balance between muscle constituents is disturbed.

An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the ␣ subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm Ϫ/Ϫ mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1␣ and Hif-2␣ protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm Ϫ/Ϫ mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2 gt/gt ) and Hif-p4h-3 Ϫ/Ϫ mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm Ϫ/Ϫ and wild-type mice, but caused higher increases in both values in the Hif-p4h-2 gt/gt mice and in hematocrit value in the Hif-p4h-3 Ϫ/Ϫ mice than in the wild-type. Hif-p4h-2 gt/gt /P4h-tm Ϫ/Ϫ double genemodified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2 gt/gt or P4h-tm Ϫ/Ϫ mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis. (Blood. 2012;120(16): 3336-3344) IntroductionErythropoiesis is a tightly controlled process, its key regulator being erythropoietin (EPO). During embryonic development most of the EPO production occurs in the liver, whereas the major EPO source in adults is the kidney, although the liver maintains a capacity for its expression. 1,2 Hypoxia-inducible transcription factor (HIF) plays a pivotal role in the regulation of the transcription of the EPO gene and numerous other hypoxia-regulated genes, including many additional genes influencing erythropoiesis. [1][2][3][4] The HIF-␣ subunit isoforms HIF-1␣ and HIF-2␣ are synthesized constitutively, and hydroxylation of 2 critical prolines generates 4-hydroxyproline residues that target HIF-␣ for rapid degradation in normoxia. [5][6][7] In hypoxia, this hydroxylation is inhibited, so that HIF-␣ escapes degradation, translocates into the nucleus, and dimerizes with HIF-␤. 5-7 HIF-1␣ is expressed in all nucleated cells, whereas HIF-2␣ expression is restricted to specific cell types, including renal interstitial cells and hepatocytes. 1,2 Renal and hepatic EPO production in adults is primarily regulated by HIF-2␣, but HIF-1␣ also plays a role in several situations. 1,2 Erythropoiesis requires iron. Hepcidin, a 25-amino-acid peptide secreted predominantly from hepatocytes, is the central regulator of iron metabolism. It down-regulates ferroportin and thus inhibits the absorption of dietary iron and the release of iron form erythrocytes and macrophages. 8,9 Hepcidin expression is lowered by hypoxia and agents that stabilize HIF-2␣ leading to increased serum EPO concentration and erythropoiesis, its regulation also involving the hemojuvelin/...

Hypoxia is a common feature in solid tumors and is associated with cancer progression. The main regulators of the hypoxic response are hypoxia-inducible transcription factors (HIFs) that guide the cellular adaptation to hypoxia by gene activation. The actual oxygen sensing is performed by HIF prolyl hydroxylases (PHDs) that under normoxic conditions mark the HIF-α subunit for degradation. Cancer progression is not regulated only by the cancer cells themselves but also by the whole tumor microenvironment, which consists of cellular and extracellular components. Hypoxic conditions also affect the stromal compartment, where stromal cells are in close contact with the cancer cells. The important function of HIF in cancer cells has been shown by many animal models and described in hundreds of reviews, but less in known about PHDs and even less PHDs in stromal cells. Here, we review hypoxic signaling in tumors, mainly in the tumor stroma, with a focus on HIFs and PHDs.