Aileen Houston

Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor

Development of the human gut microbiota commences at birth, with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date, the genetic basis of Bifidobacterium colonization and persistence remains poorly understood. Transcriptome analysis of the Bifidobacterium breve UCC2003 2.42-Mb genome in a murine colonization model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster designated “tad2003.” Mutational analysis demonstrated that the tad2003 gene cluster is essential for efficient in vivo murine gut colonization, and immunogold transmission electron microscopy confirmed the presence of Tad pili at the poles of B. breve UCC2003 cells. Conservation of the Tad pilus-encoding locus among other B. breve strains and among sequenced Bifidobacterium genomes supports the notion of a ubiquitous pili-mediated host colonization and persistence mechanism for bifidobacteria.

Immune privilege or inflammation? Insights into the Fas ligand enigma

Fas ligand (FasL) has become an enigmatic molecule: some evidence indicates that it contributes to immune privilege in tissues and tumors, whereas other data demonstrates that FasL can elicit inflammation. New findings may begin to reconcile the paradoxical effects of FasL.

Altered Mechanisms of Apoptosis in Colon Cancer: Fas Resistance and Counterattack in the Tumor‐Immune Conflict

Abstract: Fas (CD95/APO‐1) is a cell surface “death receptor” that mediates apoptosis upon engagement by its ligand, FasL. Fas‐mediated apoptosis of lymphocytes normally serves immunoregulatory roles, including tolerance acquisition, immune response termination, and maintenance of immune privilege in certain organs. Colon tumors can exploit this lymphocyte death program by expressing FasL. This may enable colon tumors to mount a “Fas counterattack” against antitumor lymphocytes, impairing antitumor immune responses. FasL‐expressing colon tumor‐derived cell lines can trigger Fas‐mediated apoptosis of cocultured T cells in vitro. FasL expressed in esophageal cancer has been significantly associated with apoptosis and depletion of tumor‐infiltrating lymphocytes (TIL) in vivo. FasL may also facilitate metastatic colonization of Fas‐sensitive organs such as the liver, by inducing apoptosis of target organ cells. Normal colonic epithelial cells express Fas and are relatively sensitive to Fas‐mediated apoptosis. By contrast, colon tumor‐derived cell lines are usually resistant to induction of Fas‐mediated apoptosis, and colon cancer cells frequently coexpress Fas and FasL. The mechanisms allowing resistance to Fasmediated apoptosis are complex, and defects have been identified at several levels of Fas signal transduction. The “Bcl‐2 rheostat” may be pitched against apoptosis in colon cancer, inasmuch as overexpression of Bcl‐2, downregulation of Bak, and mutation of Bax are common defects in colon tumors. Caspase‐1 is also downregulated in colon cancer. The high frequency of p53 mutations in late‐stage cancers may also inhibit Fas signaling. Fundamental defects in apoptosis signaling may contribute to both immuno‐ and chemoresistance in colon cancer and allow expression of FasL to counterattack antitumor lymphocytes.

Identification of TLR10 as a Key Mediator of the Inflammatory Response to Listeria monocytogenes in Intestinal Epithelial Cells and Macrophages

Listeria monocytogenes is a Gram-positive bacterium that can cause septicemia and meningitis. TLRs are central receptors of the innate immune system that drive inflammatory responses to invading microbes such as L. monocytogenes. Although intestinal epithelial cells (IECs) represent the initial point of entry used by L. monocytogenes for infection, the innate immune response to L. monocytogenes in these cells has been poorly characterized to date. The aim of this study was to determine which TLRs are involved in mediating the immune response to L. monocytogenes in IECs. We performed an RNA interference screen of TLRs 1–10 in the HT-29 IEC cell line and observed the most significant reduction in chemokine output following silencing of TLR10. This effect was also observed in the macrophage cell line THP-1. The chemokines CCL20, CCL1, and IL-8 were reduced following knockdown of TLR10. Silencing of TLR10 resulted in increased viability of L. monocytogenes in both HT-29 and THP-1 cells. TLR10 was found to be predominantly expressed intracellularly in epithelia, and activation required viable L. monocytogenes. NF-κB activation was seen to require TLR2 in addition to TLR10. Taken together, these data indicate novel roles for TLR10 in sensing pathogenic infection in both the epithelium and macrophages and have identified L. monocytogenes as a source of ligand for the orphan receptor TLR10.

Fas ligand mediates immune privilege and not inflammation in human colon cancer, irrespective of TGF-β expression

Many cancers express Fas ligand (FasL/CD95L) in vivo, and can kill lymphoid cells by Fas-mediated apoptosis in vitro. However, overexpression of recombinant FasL in murine tumour allografts revealed a potential antitumour effect of FasL, via recruitment of neutrophils. Transforming growth factor-β1 (TGF-β1) could inhibit these neutrophil-stimulatory effects of FasL. In the present study, we sought to determine directly whether FasL contributes to immune privilege or tumour rejection in human colon cancers in vivo, and whether TGF-β1 regulates FasL function. Serial tumour sections were immunostained for FasL and TGF-β1. Neutrophils and tumour infiltrating lymphocytes (TILs) were detected by immunohistochemistry for lactoferrin and CD45, respectively. Apoptotic TIL were identified by dual staining for TUNEL/CD45. FasL expression by nests of tumour cells was associated with a mean four-fold depletion of TILs (range 1.8–33-fold, n=16, P<0.001), together with a two-fold increase in TIL apoptosis (range 1.6–2.5-fold, n=14, P<0.001), relative to FasL-negative nests within the same tumours. The overall level of neutrophils present in all tumours examined was low (mean 0.3%, n=16), with FasL expression by tumour nests associated with a mean two-fold decrease in neutrophils, irrespective of TGF-β1 expression. Together, our results suggest that tumour-expressed FasL is inhibitory rather than stimulatory towards antitumour immune responses.

Modulation of p21-activated kinase 1 alters the behavior of renal cell carcinoma

The p21‐activated kinase 1 (Pak1) is a serine/threonine kinase whose activity is regulated by both Rho GTPases and AGC kinase family members. It plays a role in cytoskeletal remodeling and cell motility as well as cell proliferation, angiogenesis, tumorigenesis and metastasis. An involvement of Pak1 in renal cell carcinoma (RCC), which remains highly refractory to chemotherapy and radiotherapy, remains to be investigated. Pak1 expression, phosphorylation and kinase activity were examined in RCC cell lines and human tissue from normal and renal carcinoma. We report increased Pak1 expression and constitutive activity in the membrane and nucleus but not the cytoplasm of resected human RCC. To study a role for Pak1 in RCC, we developed 786‐0 clones that expressed either a kinase‐active Pak1L83,L86 2 different Pak1 dominant negative mutants, Pak1R299 and Pak1L83,L86,R299 or Pak1 siRNA. The expression of Pak1L83,L86 increased 786‐0 proliferation, motility and anchorage independent growth, while the dominant negative mutants and Pak1 siRNA abrogated these effects. In addition, Pak1L83,L86 conferred resistance to 5‐fluorouracil with a 40% ± 10% increase in cell viability. Conversely, Pak1L83,L86,R299, Pak1R299 and Pak1 siRNA conferred sensitivity with a 65.2% ± 5.5%, 69.2% ± 3.3% and 73.0% ± 8.4% loss in viability, respectively. Finally, Pak1 plays a role in renal tumor growth in vivo. Only 33% of mice developed tumors in the Pak1L83,L86,R299 group and no tumors developed from Pak1R299 cell challenge. Together these findings point to Pak1 as an exciting target for therapy of renal cancer, which remains highly refractory to existing treatments.

Life in the Fas lane: differential outcomes of Fas signaling

Fas, also known as CD95 or APO-1, is a member of the tumor necrosis factor/nerve growth factor superfamily. Although best characterized in terms of its apoptotic function, recent studies have identified several other cellular responses emanating from Fas. These responses include migration, invasion, inflammation, and proliferation. In this review, we focus on the diverse cellular outcomes of Fas signaling and the molecular switches identified to date that regulate its pro- and anti-apoptotic functions. Such switches occur at different levels of signal transduction, ranging from the receptor through to cross-talk with other signaling pathways. Factors identified to date including other extracellular signals, proteins recruited to the death-inducing signaling complex, and the availability of different intracellular components of signal transduction pathways. The success of therapeutically targeting Fas will require a better understanding of these pathways, as well as the regulatory mechanisms that determine cellular outcome following receptor activation.

Fas Ligand Promotes Tumor Immune Evasion of Colon Cancer In Vivo

The study of the role of Fas ligand (FasL/CD95L) in tumor immune evasion has been complicated by the discovery that FasL may trigger cytokine secretion and induce inflammation. Antisense suppression of FasL expression by colon tumor cells was used to investigate if a reduction in endogenously expressed FasL in tumors resulted in reduced tumor development and improved anti-tumor immune challenge in vivo. Downregulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immune-competent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells was associated with increased lymphocyte infiltration. Moreover, constitutively expressed FasL was not pro-inflammatory. This study indicates that upregulation of FasL expression by colon tumor cells results in an improved anti-tumor immune challenge in vivo, providing functional evidence in favor of the ‘Fas counterattack’ as a mechanism of tumor immune evasion.

Prostaglandin E2 and the EP receptors in malignancy: possible therapeutic targets?

Elevated expression of COX‐2 and increased levels of PGE2 are found in numerous cancers and are associated with tumour development and progression. Although epidemiological, clinical and preclinical studies have shown that the inhibition of PGE2 synthesis through the use of either non‐steroidal anti‐inflammatory drugs (NSAIDs) or specific COX‐2 inhibitors (COXibs) has the potential to prevent and treat malignant disease, toxicities due to inhibition of COX‐2 have limited their use. Thus, there is an urgent need for the development of strategies whereby COX‐2 activity may be reduced without inducing any side effects. The biological effects of PGE2 are mediated by signalling through four distinct E‐type prostanoid (EP) receptors – EP1, EP2, EP3 and EP4. In recent years, extensive effort has gone into elucidating the function of PGE2 and the EP receptors in health and disease, with the goal of creating selective inhibitors as a means of therapy. In this review, we focus on PGE2, and in particular on the role of the individual EP receptors and their signalling pathways in neoplastic disease. As knowledge concerning the role of the EP receptors in cancer grows, so does the potential for exploiting the EP receptors as therapeutic targets for the treatment of cancer and metastatic disease.

Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells

Fas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E2 (PGE2), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE2 increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E2-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE2 positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE2.