Neutralisation of uPA with a Monoclonal Antibody Reduces Plasmin Formation and Delays Skin Wound Healing in tPA-Deficient Mice

Background: Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds. Methodology/Principal Findings: To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficent mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficent mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure. Conclusions/Significance: Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murin

Cancer cell differentiation heterogeneity and aggressive behavior in solid tumors

The differentiation stage of tumors is a central aspect in the histopathological classification of solid malignancies. The differentiation stage is strongly associated with tumor behavior, and generally an immature tumor is more aggressive than the more differentiated counterpart. While this is common knowledge in surgical pathology, the contribution of differentiation-related gene expression and functions to tumor behavior is often overlooked in the experimental, tumor biological setting. The mechanisms by which tumor cell differentiation stages are perturbed or affected are poorly explored but have recently come into focus with the introduction.of the tumor stem cell concept. While developmental biologists view the differentiation as a unidirectional event, pathologists and tumor biologists have introduced the concept of dedifferentiation to explain phenotypic changes occurring in solid tumors. In this review we discuss the impact of the tumor cell differentiation stage as used in surgical pathology. We further discuss knowledge gained from exploring the molecular basis of the differentiation and dedifferentiation processes in neuroblastoma and breast cancer, two tumor forms where the tumor cell differentiation concept is used in the clinical diagnostic work and where the tumor stem cell theory has been applied

Prognostic impact of tumour-specific HMG-CoA reductase expression in primary breast cancer

Introduction We have previously reported that tumour-specific expression of the rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR), in the mevalonate pathway is associated with more favourable tumour parameters in breast cancer. In the present study, we examined the prognostic value of HMG-CoAR expression in a large cohort of primary breast cancer patients with long-term follow up. Methods The expression of HMG-CoAR was assessed by immunohistochemistry on tissue microarrays with tumour specimens from 498 consecutive cases of breast cancer with a median follow-up of 128 months. Kaplan Meier analysis and Cox proportional hazards modelling were used to estimate the rate of recurrence-free survival (RFS) and breast cancer specific survival (BCSS). Results In line with our previous findings, tumour-specific HMG-CoAR expression was associated with low grade (p < 0.001), small size (p = 0.007), oestrogen receptor (ER) positive (p = 0.01), low Ki-67 (p = 0.02) tumours. Patients with tumours expressing HMG-CoAR had a significantly prolonged RFS, even when adjusted for established prognostic factors (relative risk [RR] = 0.60, 95% confidence interval [CI] 0.40 to 0.92; p = 0.02). In ER-negative tumours, however, there was a trend, that was not significantly significant, towards a shorter RFS in HMG-CoAR expressing tumours. Conclusions HMG-CoAR expression is an independent predictor of a prolonged RFS in primary breast cancer. This may, however, not be true for ER-negative tumours. Further studies are needed to shed light on the value of HMG-CoAR expression as a surrogate marker of response to statin treatment, especially with respect to hormone receptor status

Transcriptional regulation in neuroblastoma cells under normoxic and hypoxic conditions

The childhood malignancy neuroblastoma develops from early cells of the sympathetic nervous system (SNS), and the tumors often produce catecholamines. Neuroblastoma cells retain several characteristics of immature sympathetic cells including the expression of a number of transcription factors normally expressed during embryogenesis. The genesis of the SNS requires the correct expression pattern of several transcription factors of the basic helix-loop-helix (bHLH) family, such as HASH-1 a pro-neuronal gene. HASH-1 expression is negatively regulated by another bHLH factor, HES-1. In these studies, we show that HASH-1 form transcription activating dimers with the ubiquitously expressed bHLH factor E2-2 in neuroblastoma cells. Furthermore, we establish that the Id proteins, which lack the basic DNA-binding domain and act as dominant negative inhibitors in the bHLH network, bind to HES-1. By dimerization with HES-1 the Id proteins may act to repress the HES-1 mediated transcriptional repression. In addition, HES-1 can alleviate the negative effect of Id proteins on bHLH factor induced transcription, by sequestration of Id. These findings reveal a novel regulatory level of the bHLH network. Solid tumors most often contain regions with impaired circulation and hypoxia. Two transcription factors of the bHLH/PAS subgroup, the hypoxia inducible factors HIF-1a and HIF-2a, are key regulators of the cellular response to oxygen deprivation. Interestingly, HIF-2a is also expressed in the developing SNS and required for catecholamine production, and HIF-2a deficient mice die with bradycardia before birth. The dual implications of HIF-2a in neuroblastoma tumors, involvement in SNS development as well as in adaptation to the tumor microenvironment , prompted us to investigate how neuroblastoma cells respond to growth under hypoxic conditions. Unexpectedly, we found that hypoxia (1% oxygen) drive the neuroblastoma cells toward an immature and neural crest-like phenotype. Several neuronal and neuroendocrine marker genes, such as chromogranin A/B, NPY, and HASH-1, were down-regulated in response to oxygen deprivation, whereas a number of genes expressed during early neural crest development were up-regulated, examplified by c-kit, Notch-1, and Id2. To further delineate the the hypoxic phenotype of human neuroblastoma cells, we have analyzed their gene expression after 72 h exposure to hypoxia employing microarray analysis harboring 35 000 clones. Almost one percent of the represented transcripts were up-regulated more than three-fold and about 0.5 % were down-regulated more than three-fold in response to hypoxia. The microarray results strenghten our view of hypoxic neuroblastoma cells as less mature. To test whether hypoxia-induced dedifferentiation is a neuroblastoma specific event or may occur also in other solid tumors, we have analyzed a panel of ductal breast carcinoma in situ. Also in these tumors, were hypoxia associated with a less mature phenotype of the tumor cells. We propose hypoxia-induced dedifferentiation as one means by which intra-tumor hypoxia drives tumor propagation

Differential HIF-1α and HIF-2α Expression in Mammary Epithelial Cells during Fat Pad Invasion, Lactation, and Involution.

The development and functional cycle of the mammary gland involves a number of processes that are caricatured by breast cancer cells during invasion and metastasis. Expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 has been associated with metastatic, poor prognosis, and high-grade breast cancers. Since hypoxia affects normal epithelial differentiation, we hypothesise that HIFs are important for normal breast epithelial development and regeneration as well as cancer initiation and progression. Here, we investigated the expression of the oxygen-sensitive HIF-alpha subunits during mouse mammary gland development, lactation, and involution. In breast epithelial cells, HIF-1α was expressed during early development, prior to cell polarisation. In contrast, expression of HIF-2α occurred later and was restricted to a subpopulation of luminal epithelial cells in the lactating gland. Mammary gland involution is a developmental stage that involves extensive tissue remodelling with cell death but survival of tissue stem/progenitor cells. At this stage, HIF-2α, but little HIF-1α, was expressed in CK14-positive epithelial cells. The temporal but differential expression of the HIF-alpha subunits during the mammary gland life cycle indicates that their expression is controlled by additional factors to hypoxia. Further functional studies of the roles of these proteins in the mammary gland and breast cancer are warranted

Hypoxia, pseudohypoxia and cellular differentiation

Tumor hypoxia correlates to aggressive disease, and while this is explained by a variety of factors, one clue to understand this phenomena was the finding that hypoxia induces a de-differentiated, stem cell-like phenotype in neuroblastoma and breast tumor cells. The hypoxia inducible transcription factors (HIFs) are regulated at the translational level by fluctuating oxygen concentrations, but emerging data reveal that both HIF-1α and HIF-2α expression can be induced by aberrantly activated growth factor signaling independently of oxygen levels. Furthermore, HIF-2α is regulated by hypoxia also at the transcriptional level in neuroblastoma and glioma cells. In cultured tumor cells, HIF-2α is stabilized at physiological oxygen concentrations followed by induced expression of classical hypoxia-driven genes, resulting in a pseudohypoxic phenotype. In addition, in neuroblastoma and glioma specimens, a small subset of HIF-2α positive, HIF-1α negative, tumor cells is found adjacent to blood vessels, i.e. in areas with presumably adequate oxygenation. These tumor niches are thus pseudohypoxic, and the HIF-2α expressing cells present immature features. We have postulated that this niche in neuroblastomas encompass the tumor stem cells. Oncogenes or tumor suppressor genes associated with pseudohypoxia are frequently mutated or deleted in the germline, implicating that the pseudohypoxic phenotype indeed is tumorigenic. In summary, the hypoxic and pseudohypoxic phenotypes of solid tumors are attractive therapeutic targets

Protein kinase Cα suppresses the expression of STC1 in MDA-MB-231 breast cancer cells

Several protein kinase C (PKC) isoforms have been shown to influence different cellular processes that may contribute to the malignancy of breast cancer cells. To obtain insight into mechanisms mediating the PKC effects, global gene expression was analyzed in MDA-MB-231 breast cancer cells in which PKCα, PKCδ or PKCε had been down-regulated with siRNA. Gene set enrichment analyses revealed that hypoxia-induced genes were enriched among genes that increased in PKCα-down-regulated cells. The STC1 mRNA, encoding stanniocalcin 1, was particularly up-regulated following depletion of PKCα and was also induced by hypoxia. Both hypoxia and PKCα down-regulation also led to increased STC1 protein levels. The results demonstrate that PKCα suppresses the expression of STC1 in breast cancer cell