Lynne Bingle

The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.

The role of macrophages in tumour growth and development is complex and multifaceted. Whilst there is limited evidence that tumour‐associated macrophages (TAMs) can be directly tumouricidal and stimulate the anti‐tumour activity of T cells, there is now contrasting evidence that tumour cells are able to block or evade the activity of TAMs at the tumour site. In some cases, tumour‐derived molecules even redirect TAM activities to promote tumour survival and growth. Indeed, evidence has emerged for a symbiotic relationship between tumour cells and TAMs, in which tumour cells attract TAMs and sustain their survival, with TAMs then responding to micro‐environmental factors in tumours such as hypoxia (low oxygen tension) by producing important mitogens as well as various growth factors and enzymes that stimulate tumour angiogenesis. This review presents evidence for the number and/or distribution of TAMs being linked to prognosis in different types of human malignancy. It also outlines the range of pro‐ and anti‐tumour functions performed by TAMs, and the novel therapies recently devised using TAMs to stimulate host immune responses or deliver therapeutic gene constructs to solid tumours. Copyright

Macrophages promote angiogenesis in human breast tumour spheroids in vivo

An in vivo model has been established to study the role of macrophages in the initiation of angiogenesis by human breast tumour spheroids in vivo. The extent of the angiogenic response induced by T47D spheroids implanted into the dorsal skinfold chamber in nude mice was measured in vivo and compared to that induced by spheroids infiltrated with human macrophages prior to implantation. Our results indicate that the presence of macrophages in spheroids resulted in at least a three-fold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. The angiogenic response measured around the spheroids, 3 days after in vivo implantation, was significantly greater in the spheroids infiltrated with macrophages. The number of vessels increased (macrophages vs no macrophages 34±1.9 vs 26±2.5, P<0.01), were shorter in length (macrophages vs no macrophages 116±4.92 vs 136±6.52, P<0.008) with an increased number of junctions (macrophages vs no macrophages 14±0.93 vs 11±1.25, P<0.025) all parameters indicative of new vessel formation. This is the first study to demonstrate a role for macrophages in the initiation of tumour angiogenesis in vivo.

The putative ovarian tumour marker gene HE4 (WFDC2), is expressed in normal tissues and undergoes complex alternative splicing to yield multiple protein isoforms

The whey acidic protein (WAP) domain is a conserved motif, containing eight cysteines found in a characteristic 4-disulphide core arrangement, that is present in a number of otherwise unrelated proteins. WAP motifs are present in SLPI and elafin, two antiproteinases located on chromosome 20q12-13, in a locus rich in poorly characterized WAP domain proteins. One of these proteins, which contains two WAP domains, is HE4 (also known as WFDC2), originally described as an epididymis specific protein but more recently suggested to be a putative serum tumour marker for ovarian cancer. We have shown that HE4 is expressed in a number of normal human tissues outside of the male reproductive system, including regions of the respiratory tract and nasopharynx, as well as in a subset of lung tumour cell lines. Comparison of multiple HE4 cDNAs and RT–PCR products with genomic sequence allowed the elucidation of the genomic organization. These studies revealed that HE4 can undergo a complex series of alternative splicing events that can potentially yield five distinct WAP domain containing protein isoforms. These results cast doubt on the potential role of HE4 as a serum tumour marker specific for ovarian cancer and open the door to understanding the function of multiple WAP domain containing protein isoforms arising from a single gene.

Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor

Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role of TLR4 (mediating responses to LPS) and TLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 and TLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration of ADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that the TLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.

WFDC2 (HE4): a potential role in the innate immunity of the oral cavity and respiratory tract and the development of adenocarcinomas of the lung.

BackgroundThe Whey Acidic Protein domain is an evolutionarily conserved motif found in a number of proteins, the best studied of which are antiproteinases involved in the innate immune defence of multiple epithelia. We recently characterised the WFDC2 gene which encodes a two WAP domain-containing protein, initially suggested as a marker for epididymis, and showed that it is highly expressed in the lung and salivary gland. The precise location of WFDC2 protein in these sites has not been described.MethodsWe used immunohistochemistry to localise WFDC2 in normal tissues of the respiratory tract, naso- and oropharynx, as well as in chronically inflamed lung from Cystic Fibrosis and a range of pulmonary carcinomas. We have complemented these studies with molecular analysis of WFDC2 gene expression in primary human lung cell cultures.ResultsWFDC2 is expressed in some epithelial cells of the upper airways as well as in mucous cells and ducts of submucosal glands. No staining was seen in peripheral lung. Intense staining is found in major salivary glands and in minor glands of the nose, sinuses, posterior tongue and tonsil. Studies with the related protein Secretory Leukocyte Protease Inhibitor (SLPI) show that although both proteins are expressed in similar tissues, the precise cellular localisation differs. Significant increases in expression and localisation of WFDC2 are seen in patients with Cystic Fibrosis. SLPI expression was greatly reduced in the same samples. In cultures of tracheobronchial epithelial cells, expression of WFDC2 and SLPI are differentially regulated during differentiation yet WFDC2 is not induced by pro-inflammatory mediators. The majority of adenocarcinomas stain with WFDC2 whilst a significant minority of squamous, small cell and large cell carcinomas exhibit focal staining. There is no clear association with tumour grade.ConclusionWe believe that these studies support the hypothesis that WFDC2 may be a component of the innate immune defences of the lung, nasal and oral cavities and suggest that WFDC2 functions in concert with related WAP domain containing proteins in epithelial host defence. We also suggest that WFDC2 re-expression in lung carcinomas may prove to be associated with tumour type and should be studied in further detail.

In vitro study of HIF-1 activation and VEGF release by bFGF in the T47D breast cancer cell line under normoxic conditions: involvement of PI-3K/Akt and MEK1/ERK pathways.

Hypoxia‐inducible factor (HIF) is critical in the modulation of tumour angiogenesis in response to hypoxia. In the present study, the mechanisms underlying basic fibroblast growth factor (bFGF)‐induced activation of HIF‐1 and the subsequent release of vascular endothelial growth factor (VEGF) in a human breast cancer cell line (T47D) under normoxic conditions were explored. The data show that HIF‐1α expression is induced by bFGF in a dose‐ and time‐dependent fashion, while increased HIF‐1α protein expression and transactivity of HIF‐1 are due to the phosphorylation of Akt by bFGF, as indicated by application of the phosphatidylinositol 3‐kinase (PI‐3K) inhibitor LY294002. The data also show that the MEK1 (mitogen‐activated protein kinase kinase‐1)/ERK (extracellular signal‐regulated kinase) pathway is only involved in bFGF‐induced transactivity of HIF‐1, but not HIF‐1α expression, indicating roles for both the PI‐3K/Akt and the MEK1/ERK pathways in bFGF activity. In addition, the translation inhibitor cycloheximide confirmed that bFGF‐induced HIF‐1α protein expression was due to de novo protein synthesis. In contrast, p38 was not required for the expression of HIF‐1α or HIF‐1 transactivity, although significant phosphorylation of p38 was observed after bFGF treatment. Treatment of the cells with bFGF increased the amount of VEGF release, and this could be suppressed by either PD98059 or LY294002, suggesting the presence of a HIF‐1α‐dependent pathway for bFGF‐induced VEGF production. In conclusion, the PI‐3K/Akt and MEK1/ERK pathways, in a potentially independent and co‐operative fashion, can modulate HIF‐1 activation by bFGF. Further studies will pinpoint whether HIF‐1 is the transcriptional factor responsible for the increased VEGF production following bFGF treatment of breast tumour cells. Copyright

Phylogenetic and evolutionary analysis of the PLUNC gene family.

The PLUNC family of human proteins are candidate host defense proteins expressed in the upper airways. The family subdivides into short (SPLUNC) and long (LPLUNC) proteins, which contain domains predicted to be structurally similar to one or both of the domains of bactericidal/permeability‐increasing protein (BPI), respectively. In this article we use analysis of the human, mouse, and rat genomes and other sequence data to examine the relationships between the PLUNC family proteins from humans and other species, and between these proteins and members of the BPI family. We show that PLUNC family clusters exist in the mouse and rat, with the most significant diversification in the locus occurring for the short PLUNC family proteins. Clear orthologous relationships are established for the majority of the proteins, and ambiguities are identified. Completion of the prediction of the LPLUNC4 proteins reveals that these proteins contain approximately a 150‐residue insertion encoded by an additional exon. This insertion, which is predicted to be largely unstructured, replaces the structure homologous to the 40s hairpin of BPI. We show that the exon encoding this region is anomalously variable in size across the LPLUNC proteins, suggesting that this region is key to functional specificity. We further show that the mouse and human PLUNC family orthologs are evolving rapidly, which supports the hypothesis that these proteins are involved in host defense. Intriguingly, this rapid evolution between the human and mouse sequences is replaced by intense purifying selection in a large portion of the N‐terminal domain of LPLUNC4. Our data provide a basis for future functional studies of this novel protein family.

SPLUNC1 (PLUNC) is expressed in glandular tissues of the respiratory tract and in lung tumours with a glandular phenotype

Short PLUNC1 (SPLUNC1) is the founding member of a novel family of proteins (PLUNC) expressed in the upper respiratory tract that may function in host defence. It is one of the most highly expressed genes in the upper airways and the protein has been detected in sputum and nasal secretions. This study describes, for the first time, the precise cellular localization of SPLUNC1 in human tissues from the respiratory tract. Although SPLUNC1 is found in some epithelial cells of the upper airways and coats the surface epithelial cell lining of the major airways, the most significant site of protein localization is in mucous cells and ducts of submucosal glands. Intense staining is also seen in minor glands of the nose, sinuses, posterior tongue and tonsil, suggesting that the protein is secreted into mucoid secretions of these tissues, where it probably functions in host defence. No staining was seen in peripheral lung tissue. As SPLUNC1 has been suggested to be a novel lung cancer marker, a limited panel of lung cancers was also studied. The findings suggest that SPLUNC1 is commonly expressed in adenocarcinomas, muco‐epidermoid carcinoma, and bronchio‐alveloar carcinoma, and is absent from small‐cell carcinoma and squamous cell carcinoma. This expression pattern is consistent with the presumed phenotypic origin of these tumours and suggests that SPLUNC1 may be a useful marker for lung cancer. Copyright

Differential epithelial expression of the putative innate immune molecule SPLUNC1 in Cystic Fibrosis

IntroductionShort PLUNC1 (SPLUNC1) is the founding member of a family of proteins (PLUNCS) expressed in the upper respiratory tract and oral cavity, which may function in host defence. It is one of the most highly expressed genes in the upper airways and the protein has been detected in sputum and nasal secretions. The biology of the PLUNC family is poorly understood but in keeping with the putative function of the protein as an immune defence protein, a number of RNA and protein studies have indicated that SPLUNC1 is increased in inflammatory/infectious conditions such as Cystic Fibrosis (CF), COPD and allergic rhinitis.MethodsWe used immunohistochemistry to localise SPLUNC1 in lung tissue from patients with CF and a range of other lung diseases. We used a range of additional markers for distinct cell types to try to establish the exact site of secretion of SPLUNC1. We have complemented these studies with a molecular analysis of SPLUNC1 gene expression in primary human lung cell cultures and isolated inflammatory cell populations.ResultsIn CF, expression of SPLUNC1 is significantly elevated in diseased airways and positive staining was noted in some of the inflammatory infiltrates. The epithelium of small airways of CF lung exhibit significantly increased SPLUNC1 staining compared to similar sized airways in non-CF lungs where staining is absent. Strong staining was also seen in mucous plugs in the airways, these included many inflammatory cells. No alveolar epithelial staining was noted in CF tissue. Airway epithelial staining did not co-localise with MUC5AC suggesting that the protein was not produced by goblet cells. Using serial sections stained with neutrophil elastase and CD68 we could not demonstrate co-localisation of SPLUNC1 with either neutrophils or macrophages/monocytes, indicating that these cells were not a source of SPLUNC1 in the airways of CF lungs. No change in staining pattern was noted in the small airways or lung parenchyma of other lung diseases studied including, COPD, emphysema or pneumonia where significant NE and CD68 staining was noted. Cultures of primary tracheobronchial epithelial cells were analysed by RT-PCR and showed that pro-inflammatory mediators did not induce expression of SPLUNC1. We have also shown that SPLUNC1 gene expression was not seen in isolated human mononuclear cells, macrophages or neutrophils.ConclusionThese studies show that SPLUNC1 is specifically and significantly increased in the small airways of lungs from patients with CF. They further suggest that it is the airway epithelium that is responsible for the increased levels of SPLUNC1 in CF and not inflammatory cells; this could be a defensive response to the infectious component of the disease.

Basic FGF augments hypoxia induced HIF-1-alpha expression and VEGF release in T47D breast cancer cells

Aim: Both hypoxia inducible factor 1 (HIF‐1) and basic fibroblast growth factor (bFGF) play important roles in tumour angiogenesis. This study was designed to clarify the cooperative effect of these two mediators in induction of vascular endothelial cell growth factor (VEGF) release from breast cancer and probe possible mechanisms involved. Methods: Release of VEGF from a breast cancer cell line (T47D) was quantitated by enzyme linked immunosorbent assay (ELISA). Expression of HIF‐1 and ERK was assayed using Western blotting. Transient transfection and dual luciferase reporter assay were used to study HIF‐1 transactivity. Results: The data showed that hypoxia induced the expression of HIF‐1&agr; protein, the transactivity of HIF‐1 and the release of VEGF. bFGF further augmented these hypoxic inductions. The PI3K pathway was required for these processes as demonstrated by application of PI3Kinase inhibitor (LY294002) or mutant construct transfections. In contrast, the MEK1 inhibitor PD98059 showed no effect on either activation of HIF‐1 or VEGF release, which is in agreement with our finding that ERK1/2 was not activated by hypoxia. Under hypoxic conditions, bFGF activated the MEK1/ERK pathway. PD98059 blocked the activation of ERK1/2 and suppressed bFGF‐induced HIF‐1 transactivity, yet the protein expression of HIF‐1&agr; or VEGF release was not affected by PD98059. Conclusion: bFGF augments hypoxia induced VEGF release mainly through the PI3K pathway and partly depending on HIF‐1 activity. Elucidation of this mechanism may provide a new target for anti‐angiogenesis in cancer therapy.