Loss of ASAP1 in mice impairs adipogenic and osteogenic differentiation of mesenchymal progenitor cells through dysregulation of FAK/Src and AKT signaling.

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT/GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells.

Spatiotemporally controlled induction of gene expression in vivo allows tracking the fate of tumor cells that traffic through the lymphatics.

Metastasis is a multistep process, during which circulating tumor cells traffic through diverse anatomical locations. Stable inducible marking of tumor cells in a manner that is tightly spatially and temporally controlled would allow tracking the contribution of cells passing through specific locations to metastatic dissemination. For example, tumor cells enter the lymphatic system and can form metastases in regional lymph nodes, but the relative contribution of tumor cells that traffic through the lymphatic system to the formation of distant metastases remains controversial. Here, we developed a novel genetic switch based on mild transient warming (TW) that allows cells to be marked in a defined spatiotemporal manner in vivo. Prior to warming, cells express only EGFP. Upon TW, the EGFP gene is excised and expression of mCherry is permanently turned on. We employed this system in an experimental pancreatic cancer model and used localized TW to induce the genetic switch in tumor cells trafficking through tumor-draining lymph nodes. Thereby we found that tumor cells disseminating via the lymphatics make a major contribution to the seeding of lung metastases. The inducible genetic marking system we have developed is a powerful tool for the tracking of metastasizing cells in vivo.

Tspan8 is expressed in breast cancer and regulates E-cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8- tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up-regulation of E-cadherin and down-regulation of Twist, p120-catenin, and ß-catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal-epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell-cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several-fold increase in EV number in cell culture and the circulation of tumour-bearing animals. We observed increased protein levels of E-cadherin and p120-catenin in these EVs; furthermore, Tspan8 and p120-catenin were co-immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Footprintless disruption of prosurvival genes in aneuploid cancer cells using CRISPR/Cas9 technology.

CRISPR/Cas9 has emerged as a powerful methodology for the targeted editing of genomic DNA sequences. Nevertheless, the intrinsic inefficiency of transfection methods required to use this technique with cultured cells requires the selection and isolation of successfully modified cells, which invariably subjects the cells to stress. Here we report a workflow that allows the isolation of genomically modified cells, even where loss of functional alleles constitutes a selective disadvantage owing to impaired ability to survive stress. Using targeted disruption of the Id1 and Id3 genes in murine B16-F10 and Ret melanoma cell lines as an example, we show that the method allows for the footprintless isolation of CRISPR/Cas9-modified aneuploid cancer cells. We also provide evidence that serial CRISPR/Cas9 modifications can occur, for example when initial homologous recombination events introduce cryptic PAM sequences, and demonstrate that multiple alleles can be successfully targeted in aneuploid cancer cells. By sequencing individual alleles we also found evidence for CRISPR/Cas9-induced transposable element insertion, albeit at a low frequency. This workflow should have broad application in the functional analysis of prosurvival gene function in cultured cells.

VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.

VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41+ cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41+ cells in the BM and a significant increase in diploid CD41+ cells, whereas the number of polyploid CD41+ cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.

Fe-doped 45S5 bioactive glass compositions impair the metabolic activity and proliferation of metastatic human breast cancer cellsin vitro.

Many kinds of human tumors, including breast carcinomas, frequently metastasize to the bone, making it prone to pathologic fractures. Surgical management of bone metastases ranges from the resection of metastases to bone repair. Current surgical methods for the repair of bone defects include the use of polymethyl methacrylate (PMMA)-based bone cements. A promising alternative material are bioactive glass (BG) particles that in addition to providing physical stability can also induce bone regeneration. Moreover, BGs doped with Fe2O3may also have a negative impact on tumor cells. Here, we tested the hypothesis that BGs can affect metastatic human breast cancer cells. To this end, we assessed the effects of different BG compositions with and without Fe2O3on metastatic human MDA-MB-231 breast cancer cellsin vitro. We found that all BGs tested impaired the viability and proliferation of breast cancer cells in a concentration-dependent manner. The anti-proliferative effects inversely correlated with BG particle size, and were in general less pronounced in mesenchymal stromal cells (MSCs) that served as a control. Moreover, Fe2O3-doped BGs were more potent inhibitors of tumor cell proliferation and metabolic activity than Fe2O3-free BG. Our data therefore indicate that BGs can affect human breast cancer cells more strongly than MSCs, and suggest that the presence of Fe2O3can potentiate anti-proliferative and anti-metabolic effects of BGs. Fe2O3-doped BGs thus have the potential to be used for the surgical management of metastatic bone lesions, and may in addition to their regenerative properties also allow the local control of bone metastases.

Comparison of Decellularized Human Dermal Scaffolds versus Bovine Collagen/Elastin Matrices for Engineering of Soft-Tissue Flaps.

BACKGROUND: Free flap-based soft-tissue reconstruction comes at the price of donor-site morbidity. The arteriovenous loop (AVL) technique can overcome this issue by allowing for the de novo generation of axially vascularized soft-tissue flaps from vein grafts embedded into different matrices. Application of the AVL technique has been limited by insufficient long-term volume retention and poor tissue stability. The authors investigated the suitability of a novel human dermal scaffold to improve volume retention and tissue stability. METHODS: AVLs were created in 28 immunocompetent rats and embedded in either decellularized human dermal scaffolds (experimental group, n = 14) (Epiflex) or bovine collagen/elastin matrices (control group, n = 14) (MatriDerm) in subcutaneous polytetrafluoroethylene chambers. The weight and volume of engineered tissues, the extent of angiogenesis, and the proportion of proliferating cells were compared between groups on postoperative days (PODs) 21 and 28 by means of immunohistochemistry and micro-computed tomography. RESULTS: On POD 28, both groups displayed homogeneous microvascular networks on histopathology and micro-computed tomography. Mean microvessel counts and surface areas and the percentage of proliferating cells did not differ between the groups. However, the experimental human scaffold group displayed significantly smaller volume loss and significantly less tissue degradation compared with bovine matrix controls (volume retention, 102% ± 5% versus 27% ± 7% on POD 21, and 79% ± 12% versus 12% ± 7% on POD 28, respectively; P < 0.0001). CONCLUSION: Compared with bovine matrices, decellularized human scaffolds allow for superior volume retention and tissue stability of de novo engineered soft-tissue AVL flaps in rats. CLINICAL RELEVANCE STATEMENT: AVLs allow for the de novo generation of vascularized soft-tissue flaps. However, insufficient long-term volume retention is still an issue. The authors' study shows that decellularized human matrices guarantee superior volume stability of de novo grown soft-tissue flaps in rats.

In Vivo Engineering and Transplantation of Axially Vascularized and Epithelialized Flaps in Rats.

The arteriovenous loop (AVL) model allows the in vivo engineering of axially vascularized flaps, the so-called AVL flaps. Although AVL flaps can be transplanted microsurgically to cover tissue defects, they lack an epithelial layer on the surface. Therefore, the objective of this study was to engineer axially vascularized AVL flaps with an accompanying epithelial layer for local defect reconstruction. In this study, AVLs were established in 20 male Lewis rats. Minimally invasive injection of keratinocytes onto the surface of the AVL flaps was performed on postoperative day (POD) 21. AVL flaps were explanted from 12 rats on POD 24 or POD 30, then the epithelium formed by the keratinocytes on the surface of the flaps was evaluated using immunofluorescence staining. In six other rats, the AVL flap was locally transposed to cover a critical defect in the rats' leg on POD 30 and explanted for analysis on POD 40. In two control rats, sodium chloride was applied instead of keratinocytes. These control flaps were also transplanted on POD 30 and explanted on POD 40. Our results revealed that 3 days after keratinocyte application, a loose single-layered epithelium was observed histologically on the AVL flaps surface, whereas after 9 days, a multilayered and structured epithelium had grown. The epithelium on the transplanted AVL flaps showed its physiological differentiation when being exposed to an air-liquid interface. Histologically, a layered epithelium identical to the rats' regular skin was formed. In the sodium chloride control group, no epithelium had been grown. This study clearly demonstrates that axially vascularized AVL flaps can be processed in the subcutaneous chamber by minimally invasive injection of keratinocytes. Thus, AVL flaps with an intact epithelial layer were engineered and could be successfully transplanted for local defect coverage in a small animal model.

Delphinidin is a novel inhibitor of lymphangiogenesis but promotes mammary tumor growth and metastasis formation in syngeneic experimental rats.

We have recently demonstrated that the anthocyanidin delphinidin (DEL), one of the most abundant dietary flavonoids, inhibits activation of ErbB and vascular endothelial growth factor receptor family members. These receptors play crucial roles in the context of tumor progression and the outgrowth of blood and lymphatic vessels. Here, we have developed an improved chemical synthesis for DEL in order to study the effects of the aglycon and its degradation product gallic acid (GA) on endothelial and tumor cells in vitro and in vivo. We found that DEL blocked the proliferation in vitro of primary human blood and lymphatic endothelial cells as well as human HT29 colon and rat MT-450 mammary carcinoma cells in a dose-dependent manner. In contrast, its degradation product GA had little effect. At higher concentrations, DEL induced apoptosis of endothelial and tumor cells. Furthermore, DEL potently blocked the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In the MT-450 rat syngeneic breast tumor model, it also significantly reduced angiogenesis and tumor-induced lymphangiogenesis when administered in vivo. These data reveal DEL to be a novel antilymphangiogenesis reagent. Surprisingly, however, the application of DEL unexpectedly promoted tumor growth and metastasis in the MT-450 tumor model, suggesting that the antiproliferative effect of DEL on cultured cells does not necessarily reflect the response of tumors to this anthocyanidin in vivo. Furthermore, while DEL may have utility as a cancer chemopreventative agent, its ability to promote tumor growth once a neoplasm develops also needs to be taken into consideration.

Tumor-initiating properties of breast cancer and melanoma cells in vivo are not invariably reflected by spheroid formation in vitro, but can be increased by long-term culturing as adherent monolayers.

Cancer stem cells (CSCs) have been studied intensively in recent years due to their potential importance for understanding neoplastic disease and the design of antitumor therapies. A number of properties attributed to CSCs have been used to define the CSC population, the most important of which is the ability to initiate reproducibly the growth of tumors in vivo. Other assays such as spheroid formation, expression of particular markers and label retention are also used for defining CSCs, although the degree to which these assays invariably reflect the ability to form tumors in vivo remains to be carefully evaluated. Given the importance of correctly defining and isolating CSCs if valid conclusions about their characteristics are to be made, we used syngeneic animal models to compare these different assays. In standard spheroid assays, cell aggregation rather than spheroid growth from single cell suspensions ensued, but aggregation was circumvented by the inclusion of methylcellulose in the medium. Label-retaining subpopulations did not reliably exhibit an enhanced ability to form spheroids and were enriched for senescent cells. Spheroid-forming ability was found to correspond to expression of established CSC markers, although not invariably. Furthermore, spheroid-forming ability was not always reflected in tumor-initiating properties in vivo. Long-term culture of primary mammary tumor cells as adherent monolayers increased their tumor-initiating ability in vivo. This increase was attenuated when the cells were subsequently cultivated as spheroids. Together these data indicate that assays that are widely used to define CSC subpopulations do not invariably reflect tumor-initiating properties in vivo.

CD24 interacts with and promotes the activity of c-src within lipid rafts in breast cancer cells, thereby increasing integrin-dependent adhesion.

Expression of the glycosylphosphatidylinositol-anchored membrane protein CD24 correlates with a poor prognosis for many human cancers, and in experimental tumors can promote metastasis. However, the mechanism by which CD24 contributes to tumor progression remains unclear. Here we report that in MTLy breast cancer cells CD24 interacts with and augments the kinase activity of c-src, a protein strongly implicated in promoting invasion and metastasis. This occurs within and is dependent upon intact lipid rafts. CD24-augmented c-src kinase activity increased formation of focal adhesion complexes, accelerated phosphorylation of FAK and paxillin and consequently enhanced integrin-mediated adhesion. Loss and gain of function approaches showed that c-src activity is necessary and sufficient to mediate the effects of CD24 on integrin-dependent adhesion and cell spreading, as well as on invasion. Together these results indicate that c-src is a CD24-activated mediator that promotes integrin-mediated adhesion and invasion, and suggest a mechanism by which CD24 might contribute to tumor progression through stimulating the activity of c-src or another member of the Src family.

Increased Circulating Osteopontin Levels Promote Primary Tumour Growth, but Do Not Induce Metastasis in Melanoma.

Osteopontin (OPN) is a phosphoprotein with diverse functions in various physiological and pathological processes. OPN expression is increased in multiple cancers, and OPN within tumour tissue has been shown to promote key stages of cancer development. OPN levels are also elevated in the circulation of cancer patients, which in some cases has been correlated with enhanced metastatic propensity and poor prognosis. However, the precise impact of circulating OPN (cOPN) on tumour growth and progression remains insufficiently understood. To examine the role of cOPN, we used a melanoma model, in which we stably increased the levels of cOPN through adeno-associated virus-mediated transduction. We found that increased cOPN promoted the growth of primary tumours, but did not significantly alter the spontaneous metastasis of melanoma cells to the lymph nodes or lungs, despite an increase in the expression of multiple factors linked to tumour progression. To assess whether cOPN has a role at later stages of metastasis formation, we employed an experimental metastasis model, but again could not detect any increase in pulmonary metastasis in animals with elevated levels of cOPN. These results demonstrate that increased levels of OPN in the circulation play distinct roles during different stages of melanoma progression.

Ketogenic diet does not promote triple-negative and luminal mammary tumor growth and metastasis in experimental mice.

Ketogenic diets (KDs) can improve the well-being and quality of life of breast cancer patients. However, data on the effects of KDs on mammary tumors are inconclusive, and the influence of KDs on metastasis in general remains to be investigated. We therefore assessed the impact of a KD on growth and metastasis of triple negative murine 4T1 mammary tumors, and on the progression of luminal breast tumors in an autochthonous MMTV-PyMT mouse model. We found that KD did not influence the metastasis of 4T1 and MMTV-PyMT mammary tumors, but impaired 4T1 tumor cell proliferation in vivo, and also temporarily reduced 4T1 primary tumor growth. Notably, the ketogenic ratio (the mass of dietary fat in relation to the mass of dietary carbohydrates and protein) that is needed to induce robust ketosis was twice as high in mice as compared to humans. Surprisingly, only female but not male mice responded to KD with a sustained increase in blood ß-hydroxybutyrate levels. Together, our data show that ketosis does not foster primary tumor growth and metastasis, suggesting that KDs can be safely applied in the context of luminal breast cancer, and may even be advantageous for patients with triple negative tumors. Furthermore, our data indicate that when performing experiments with KDs in mice, the ketogenic ratio needed to induce ketosis must be verified, and the sex of the mice should also be taken into account.

Acellular Human Placenta Small-Diameter Vessels as a Favorable Source of Super-Microsurgical Vascular Replacements: A Proof of Concept.

In this study, we aimed to evaluate the human placenta as a source of blood vessels that can be harvested for vascular graft fabrication in the submillimeter range. Our approach included graft modification to prevent thrombotic events. Submillimeter arterial grafts harvested from the human placenta were decellularized and chemically crosslinked to heparin. Graft performance was evaluated using a microsurgical arteriovenous loop (AVL) model in Lewis rats. Specimens were evaluated through hematoxylin-eosin and CD31 staining of histological sections to analyze host cell immigration and vascular remodeling. Graft patency was determined 3 weeks after implantation using a vascular patency test, histology, and micro-computed tomography. A total of 14 human placenta submillimeter vessel grafts were successfully decellularized and implanted into AVLs in rats. An appropriate inner diameter to graft length ratio of 0.81 ± 0.16 mm to 7.72 ± 3.20 mm was achieved in all animals. Grafts were left in situ for a mean of 24 ± 4 days. Decellularized human placental grafts had an overall patency rate of 71% and elicited no apparent immunological responses. Histological staining revealed host cell immigration into the graft and re-endothelialization of the vessel luminal surface. This study demonstrates that decellularized vascular grafts from the human placenta have the potential to serve as super-microsurgical vascular replacements.

Fat Grafts Show Higher Hypoxia, Angiogenesis, Adipocyte Proliferation, and Macrophage Infiltration than Flaps in a Pilot Mouse Study.

BACKGROUND: Over 137,000 breast reconstructions are performed annually by American Society of Plastic Surgeons (ASPS) members. Vascularized flaps and avascular lipofilling each account for over 33,000 autologous reconstructions. Although clinical and experimental observations suggest biologic differences with diverging effects on locoregional tumor control, comparative animal models are lacking. The authors standardized existing techniques in immunocompetent mice, laying the foundation for in vivo models of autologous breast reconstruction combinable with orthotopic tumor implantations. METHODS: Twenty-five groin flaps and 39 fat grafts were transferred in female BALB/c-mice. Adipocytes were tracked via Hoechst-Calcein-DiI staining ( n = 2 per group), and postoperative volume retentions were compared via magnetic resonance imaging ( n = 3 per group) on days 1, 11, 21, and 31. Proliferation indices, microvessel densities, tissue hypoxia, and macrophage infiltrates were compared via Ki67, CD31, pimonidazole, and hematoxylin-eosin staining on days 5, 10, 15, 20, and 30 ( n = 4 per group). RESULTS: Viable adipocytes were present in both groups. Graft volumes plateaued at 42.7 ± 1.2% versus 81.8 ± 4.0% of flaps ( P < 0.001). Initially, grafts contained more hypoxic cells (day 5: 15.192 ± 1.249 versus 1.157 ± 192; P < 0.001), followed by higher proliferation (day 15: 25.2 ± 1.0% versus 0.0 ± 0.0%; P < 0.001), higher microvessel numbers (day 30: 307.0 ± 13.2 versus 178.0 ± 10.6; P < 0.001), and more pronounced macrophage infiltrates (graded 3 versus 2; P < 0.01). CONCLUSION: This comparative murine pilot study of vascularized flaps versus avascular lipofilling suggests differences in volume retention, proliferation, angiogenesis, hypoxia, and inflammation. CLINICAL RELEVANCE STATEMENT: The biological differences of fat grafting versus flap transfer are not fully understood because no single comparative experimental model has been established to date. The authors present the first comparative small animal model of both techniques, which will allow the gaining of deeper insights into their biological effects.

Discovery of a novel tumour metastasis-promoting gene, NVM-1.

We have previously reported that over-expression of a panel of 119 genes correlates with the metastatic potential of pancreatic carcinoma cells. We sought to identify and functionally characterize candidate tumour metastasis promoting genes among this library using a secondary phenotype-assisted screen. Here we report the discovery of the metastasis-promoting function of a hitherto not characterized gene located on chromosome 14 (ORF138), which we have named 'novel metastasis-promoting gene 1' (NVM-1). The NVM-1 transcript is extensively alternatively spliced, is expressed endogenously in a number of different tissues, and is strongly over-expressed at the protein level in a variety of human tumour types. Importantly, NVM-1 expression stimulates the migratory and invasive behaviour of tumour cells and promotes metastasis formation in experimental animals in vivo. Up-regulation of FMNL2 and MT1E and down-regulation of TIMP4 and MHC-I is observed as a consequence of NVM-1 expression. Together these data identify NVM-1 as a gene that is functionally involved in tumour metastasis, and suggest that NVM-1 may constitute a promising therapeutic target for inhibition of tumour metastasis.

Sulfated hyaluronic acid inhibits the hyaluronidase CEMIP and regulates the HA metabolism, proliferation and differentiation of fibroblasts.

Hyaluronan (HA) is an extracellular matrix component that regulates a variety of physiological and pathological processes. The function of HA depends both on its overall amount and on its size, properties that are controlled by HA synthesizing and degrading enzymes. The lack of inhibitors that can specifically block individual HA degrading enzymes has hampered attempts to understand the contribution of individual hyaluronidases to different physiological and pathological processes. CEMIP is a recently discovered hyaluronidase that cleaves HA through mechanisms and under conditions that are distinct from those of other hyaluronidases such as HYAL1 or HYAL2. The role of its hyaluronidase activity in physiology and disease is poorly understood. Here, we characterized a series of sulfated HA derivatives (sHA) with different sizes and degrees of sulfation for their ability to inhibit specific hyaluronidases. We found that highly sulfated sHA derivatives potently inhibited CEMIP hyaluronidase activity. One of these compounds, designated here as sHA3.7, was characterized further and shown to inhibit CEMIP with considerable selectivity over other hyaluronidases. Inhibition of CEMIP with sHA3.7 in fibroblasts, which are the main producers of HA in the interstitial matrix, increased the cellular levels of total and high molecular weight HA, while decreasing the fraction of low molecular weight HA fragments. Genetic deletion of CEMIP in mouse embryonic fibroblasts (MEFs) produced analogous results and confirmed that the effects of sHA3.7 on HA levels were mediated by CEMIP inhibition. Importantly, both CEMIP deletion and its inhibition by sHA3.7 suppressed fibroblast proliferation, while promoting differentiation into myofibroblasts, as reflected in a lack of CEMIP in myofibroblasts within skin wounds in experimental mice. By contrast, adipogenic and osteogenic differentiation were attenuated upon CEMIP loss or inhibition. Our results demonstrate the importance of CEMIP for the HA metabolism, proliferation and differentiation of fibroblasts, and suggest that inhibition of CEMIP with sulfated HA derivatives such as sHA3.7 has potential utility in pathological conditions that are dependent on CEMIP function.

Loss of ASAP1 in the MMTV-PyMT model of luminal breast cancer activates AKT, accelerates tumorigenesis, and promotes metastasis.

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis in a variety of cancers, and can promote cell migration, invasion and metastasis. Although amplification and expression of ASAP1 has been associated with poor survival in breast cancer, we found that in the autochthonous MMTV-PyMT model of luminal breast cancer, ablation of ASAP1 resulted in an earlier onset of tumor initiation and increased metastasis. This was due to tumor cell-intrinsic effects of ASAP1 deletion, as ASAP1 deficiency in tumor, but not in stromal cells was sufficient to replicate the enhanced tumorigenicity and metastasis observed in the ASAP1-null MMTV-PyMT mice. Loss of ASAP1 in MMTV-PyMT mice had no effect on proliferation, apoptosis, angiogenesis or immune cell infiltration, but enhanced mammary gland hyperplasia and tumor cell invasion, indicating that ASAP1 can accelerate tumor initiation and promote dissemination. Mechanistically, these effects were associated with a potent activation of AKT. Importantly, lower ASAP1 levels correlated with poor prognosis and enhanced AKT activation in human ER+/luminal breast tumors, validating our findings in the MMTV-PyMT mouse model for this subtype of breast cancer. Taken together, our findings reveal that ASAP1 can have distinct functions in different tumor types and demonstrate a tumor suppressive activity for ASAP1 in luminal breast cancer.

Do all roads lead to Rome? Routes to metastasis development.

Metastasis, the life-threatening aspect of cancer, is a systemic disease process. Considerable progress has been made in recent years regarding how tumor cells circulating in the blood and lymphatic systems interact with and extravasate into secondary sites, and what determines whether these disseminated tumors cells survive, remain dormant or go on to form macrometastases. New insights into the routes that tumor cells take once leaving the primary tumor have emerged. Novel concepts regarding early seeding of metastases coupled to parallel progression, self-seeding of primary tumors by circulating tumor cells and the induction of premetastatic niches in distant organs by primary tumors have come to the fore. The perceived role of the lymphatic system in determining patterns of metastasis formation in distant organs has been reassessed. Together these new insights have the potential to offer new therapeutic options. In particular, the regulation of tumor cell dormancy emerges as a key event in metastasis formation, and therapeutic control of dormancy holds the promise of rendering cancer a chronic rather than life-threatening disease.

LiCl induces TNF-α and FasL production, thereby stimulating apoptosis in cancer cells.

BACKGROUND: The incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours. RESULTS: Here we show that LiCl induces apoptosis of tumour cells both in vitro and in vivo. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-α and FasL expression. Inhibition of TNF-α induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth. CONCLUSIONS: Induction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-α and FasL.Key words: LiCl, TNF-α, FasL, apoptosis, GSK-3, FasL.