Emilie Flaberg

Molecular classification of mucoepidermoid carcinomas—Prognostic significance of the MECT1–MAML2 fusion oncogene

Mucoepidermoid carcinomas (MECs) of the salivary and bronchial glands are characterized by a recurrent t(11;19)(q21;p13) translocation resulting in a MECT1–MAML2 fusion in which the CREB‐binding domain of the CREB coactivator MECT1 (also known as CRTC1, TORC1 or WAMTP1) is fused to the transactivation domain of the Notch coactivator MAML2. To gain further insights into the molecular pathogenesis of MECs, we cytogenetically and molecularly characterized a series of 29 MECs. A t(11;19) and/or an MECT1–MAML2 fusion was detected in more than 55% of the tumors. Several cases with cryptic rearrangements that resulted in gene fusions were detected. In fusion‐negative MECs, the most common aberration was a single or multiple trisomies. Western blot and immunohistochemical studies demonstrated that the MECT1–MAML2 fusion protein was expressed in all MEC‐specific cell types. In addition, cotransfection experiments showed that the fusion protein colocalized with CREB in homogeneously distributed nuclear granules. Analyses of potential downstream targets of the fusion revealed differential expression of the cAMP/CREB (FLT1 and NR4A2) and Notch (HES1 and HES5) target genes in fusion‐positive and fusion‐negative MECs. Moreover, clinical follow‐up studies revealed that fusion‐positive patients had a significantly lower risk of local recurrence, metastases, or tumor‐related death compared to fusion‐negative patients (P = 0.0012). When considering tumor‐related deaths only, the estimated median survival for fusion‐positive patients was greater than 10 years compared to 1.6 years for fusion‐negative patients. These findings suggest that molecularly classifying MECs on the basis of an MECT1–MAML2 fusion is histopathologically and clinically relevant and that the fusion is a useful marker in predicting the biological behavior of MECs.

Symbionts as major modulators of insect health: Lactic Acid Bacteria and honeybees

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.

Filamin B deficiency in mice results in skeletal malformations and impaired microvascular development.

Mutations in filamin B (FLNB), a gene encoding a cytoplasmic actin-binding protein, have been found in human skeletal disorders, including boomerang dysplasia, spondylocarpotarsal syndrome, Larsen syndrome, and atelosteogenesis phenotypes I and III. To examine the role of FLNB in vivo, we generated mice with a targeted disruption of Flnb. Fewer than 3% of homozygous embryos reached term, indicating that Flnb is important in embryonic development. Heterozygous mutant mice were indistinguishable from their wild-type siblings. Flnb was ubiquitously expressed; strong expression was found in endothelial cells and chondrocytes. Flnb-deficient fibroblasts exhibited more disorganized formation of actin filaments and reduced ability to migrate compared with wild-type controls. Flnb-deficient embryos exhibited impaired development of the microvasculature and skeletal system. The few Flnb-deficient mice that were born were very small and had severe skeletal malformations, including scoliotic and kyphotic spines, lack of intervertebral discs, fusion of vertebral bodies, and reduced hyaline matrix in extremities, thorax, and vertebrae. These mice died or had to be euthanized before 4 weeks of age. Thus, the phenotypes of Flnb-deficient mice closely resemble those of human skeletal disorders with mutations in FLNB.

High levels of thioredoxin reductase 1 modulate drug-specific cytotoxic efficacy.

The selenoprotein thioredoxin reductase 1 (TrxR1) is currently recognized as a plausible anticancer drug target. Here we analyzed the effects of TrxR1 targeting in the human A549 lung carcinoma cell line, having a very high basal TrxR1 expression. We determined the total cellular TrxR activity to be 271.4 +/- 39.5 nmol min(-1) per milligram of total protein, which by far exceeded the total thioredoxin activity (39.2 +/- 3.5 nmol min(-1) per milligram of total protein). Knocking down TrxR1 by approx 90% using siRNA gave only a slight effect on cell growth, irrespective of concurrent glutathione depletion (> or = 98% decrease), and no increase in cell death or distorted cell cycle phase distributions. This apparent lack of phenotype could probably be explained by Trx functions being maintained by the remaining TrxR1 activity. TrxR1 knockdown nonetheless yielded drug-specific modulation of cytotoxic efficacy in response to various chemotherapeutic agents. No changes in response upon exposure to auranofin or juglone were seen after TrxR1 knockdown, whereas sensitivity to 1-chloro-2,4-dinitrobenzene or menadione became markedly increased. In contrast, a virtually complete resistance to cisplatin using concentrations up to 20 microM appeared upon TrxR1 knockdown. The results suggest that high overexpression of TrxR has an impact not necessarily linked to Trx function that nonetheless modulates drug-specific cytotoxic responses.

Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded autologous or allogeneic natural killer (NK) cells mismatched for MHC class I molecules and inhibitory NK receptors. NK cell–based immunoadjuvant therapies are carried out in combination with standard chemotherapeutic protocols. In the presented study, we characterized the effect of 28 frequently used chemotherapeutic agents on the capacity of NK cells to kill target cells. We found that treatment of NK cells with the drugs vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, bortezomib, and MG-132 effectively inhibited NK cell–mediated killing without affecting the viability of NK cells. On the other hand, the following drugs permitted efficient NK cell–mediated killing even at concentrations comparable with or higher than the maximally achieved therapeutic concentration in vivo in humans: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine. [Mol Cancer Ther 2007;6(2):644–54]

High-throughput live-cell imaging reveals differential inhibition of tumor cell proliferation by human fibroblasts

Increasing evidence indicates that cancer development requires changes both in the precancerous cells and in their microenvironment. To study one aspect of the microenvironmental control, we departed from Michael Stokers observation (Stroker et al, J Cell Sci 1966;1:297–310) that normal fibroblasts can inhibit the growth of admixed cancer cells (neighbour suppression). We have developed a high‐throughput microscopy and image analysis system permitting the examination of live mixed cell cultures growing on 384‐well plates, at the single cell level and over time. We have tested the effect of 107 samples of low passage number (<5) primary human fibroblasts from pediatric and adult donors, on the growth of six human tumor cell lines. Three of the lines were derived from prostate carcinomas, two from lung carcinomas and one was an EBV‐transformed lymphoblastoid line. Labeled tumor cells were grown in the presence of unlabeled fibroblasts. The majority of the tested fibroblasts inhibited the proliferation of the tumor cells, compared to the control cultures where labeled tumor cells were co‐cultured with unlabeled tumor cells. The proliferation inhibiting effect of the fibroblasts differed depending on their site of origin and the age of the donor. Inhibition required direct cell contact. Mouse 3T3 fibroblasts inhibited the growth of SV40‐transformed 3T3 cells and human tumor cells, showing that the inhibitory effect could prevail across the species barrier. Our high‐throughput system allows the quantitative analysis of the inhibitory effect of fibroblasts on the population level and the exploration of differences depending on the source of the normal cells.

Effect of frequently used chemotherapeutic drugs on cytotoxic activity of human cytotoxic T-lymphocytes.

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytotoxic T-lymphocytes (CTL). It is important to consider the drug-induced effects when chemotherapeutic regimens and CTL-mediated immunotherapy is planned to be used in parallel. In this study, we characterized the effect of 29 frequently used chemotherapeutic agents on the cytotoxic activity of autologous and allogeneic CTLs. We found that treatment of CTLs with the following drugs: docetaxel, vincristine, chlorambucil, mitomycin C, oxaliplatin, doxorubicin, and bleomycin effectively inhibited CTL-mediated killing, without affecting their viability. On the other hand, the following drugs enhanced or permitted efficient CTL-mediated killing in vitro at concentrations comparable with the maximally achieved therapeutic concentration in vivo in humans: daunorubicin, prednisolone, vinorelbine, cisplatin, methotrexate, hydroxyurea, cytarabine, cyclophosphamide, topotecan, epirubicin, fluorouracil, carboplatin, asparaginase, 6-mercaptopurine, and bortezomib. Our results could potentially be used in the future to design new CTL-based adjuvant immunotherapy protocols.

Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

Cyclooxygenase-2 (COX-2) expression is induced by mitogenic and proinflammatory factors. Its overexpression plays a causal role in inflammation and tumorigenesis. COX-2 expression is tightly regulated, but the mechanisms are largely unclear. Here we show the control of COX-2 expression by an endogenous tryptophan metabolite, 5-methoxytryptophan (5-MTP). By using comparative metabolomic analysis and enzyme-immunoassay, our results reveal that normal fibroblasts produce and release 5-MTP into the extracellular milieu whereas A549 and other cancer cells were defective in 5-MTP production. 5-MTP was synthesized from l-tryptophan via tryptophan hydroxylase-1 and hydroxyindole O-methyltransferase. 5-MTP blocked cancer cell COX-2 overexpression and suppressed A549 migration and invasion. Furthermore, i.p. infusion of 5-MTP reduced tumor growth and cancer metastasis in a murine xenograft tumor model. We conclude that 5-MTP synthesis represents a mechanism for endogenous control of COX-2 overexpression and is a valuable lead for new anti-cancer and anti-inflammatory drug development.

Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent

Significance Normal microenvironments can restrict cancer development and progression. Inhibition of tumor cell growth and motility by normal fibroblasts is one measurable manifestation of this multicomponential control. Here we show that inhibition withstands formalin fixation and can be augmented by the addition of conditioned medium derived from live cultures of tumor cells confronting the stromal fibroblasts. We describe a number of molecules involved in this process. This study lays the foundation for further mechanistic studies of this important phenomenon and its contribution to possible dormancy and the tumor’s resistance to therapy. Normal human and murine fibroblasts can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. We showed previously that effective inhibition requires formation of a morphologically intact fibroblast monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the factors responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the fibroblasts, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble factors secreted by fibroblasts when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from fibroblasts alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show up-regulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of growth differentiation factor 15, dickkopf-related protein 1, endothelial-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.

The architecture of fibroblast monolayers of different origin differentially influences tumor cell growth.

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when co‐cultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. It requires direct cell‐to‐cell contact and is not transferable with supernatant. Here, we show that effective inhibition also requires the formation of a morphologically intact fibroblast monolayer before the seeding of the tumor cells. Interference with the formation of the monolayer impairs the inhibition. Subclones of TERT‐immortalized fibroblasts were selected on the basis of differences in the growth pattern and related inhibitory activity. Whereas the well‐organized “whirly” (WH) growth pattern was associated with strong inhibition, the disorganized “crossy” (CR) growth pattern was linked to reduced inhibition. Time lapse imaging of tumor‐fibroblast co‐cultures using extended field live cell microscopy revealed that fibroblast monolayers with growth inhibitory capacity also reduced the motility of the tumor cells whereas noninhibitory monolayers had no effect on tumor cell motility. Gene expression pattern of two isogenic pairs of fibroblasts, WH and CR subclones of the TERT immortalized line (inhibitory, and less inhibitory subsequently) and freshly explanted skin (inhibitory) and hernia (noninhibitory) fibroblasts derived from the same patient, identified a set of genes that co‐segregated with the inhibitory phenotype. This suggests that our model system may reveal molecular mechanisms involved in contact‐mediated microenvironmental surveillance that may protect the organism from the outgrowth of disseminated tumor cells.