Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells

<p>Abstract</p> <p>Background</p> <p>Recently, periostin (<it>POSTN</it>), a gene encoding a protein with similarity to the fasciclin family and involved in cell survival and angiogenesis, has emerged as a promising marker for tumor progression in various types of human cancers. There is some controversy regarding both <it>POSTN </it>expression levels and the nature of periostin-producing cells within tumors. In this study, we used quantitative RT-PCR to assess periostin gene expression in normal tissues, primary cell cultures, tumor tissues and tumor cell lines.</p> <p>Results</p> <p>Periostin expression levels are highly variable in both normal tissues and tumors and strong <it>POSTN </it>overexpression is mostly detected in tumors from pancreas and liver. <it>POSTN </it>is not expressed in blood cancers. In melanoma samples, average periostin expression is not increased in primary tumors whereas <it>POSTN </it>overexpression was detected in about 60% of melanoma metastatic tumors in the liver or lymph nodes. Identification of the cellular source of periostin production in melanoma metastases -cancer cells or stroma- was assessed by comparing periostin expression in 23 newly-established melanoma cell lines and matched tumors. In contrast to the reduction by more than 99% of <it>COL6A3 </it>stromal marker mRNA in all cell lines, significant <it>POSTN </it>transcription was maintained in some melanoma cell lines, suggesting that both stromal cells and melanoma cells express periostin. The high level of periostin expression in primary cultures of skin fibroblasts suggests that fibroblasts may contribute for a large part to periostin production in melanoma-associated stroma. On the other hand, periostin expression in melanoma cells is probably acquired during the tumorigenic process as 1) normal melanocytes do not express <it>POSTN </it>and 2) melanoma cells from distinct metastases of the same patient were associated with very different levels of periostin expression.</p> <p>Conclusion</p> <p>Our comparative analysis suggests that, although periostin overexpression is clearly detected in some cancers, it is not a general feature of tumors. In melanoma, our study identifies both stromal and melanoma cells as sources of periostin production and correlates <it>POSTN </it>expression levels with increased primary tumor thickness and metastatic process development.</p

The odyssey of telomerase in regulating cellular responses to cytokines and reactive oxygen species

During the multistep tumorigenic process, cancer cells progressively acquire properties that include an unlimited replicative potential, invasion, sustained proliferative signaling, metastasis and resistance to cell death. In addition to its well-established role in telomere synthesis, telomerase was reported to exert non-canonical functions that may promote several abilities of cancer cells, notably by lowering reactive oxygen species (ROS) levels, modulating chromatin properties and acting as transcriptional cofactor in Wnt/β-catenin signaling pathway. The aim of this thesis was to investigate possible non-canonical functions of telomerase in regulating human fibroblasts responses to cytokines, namely Tumor Necrosis Factor-α (TNF-α) pro-inflammatory cytokine and Transforming Growth Factor-β1 (TGF-β1). ROS have been involved in a variety of cellular events downstream of TNF-α binding to its receptor, including mitogen-activated protein kinase (MAPK) activation, apoptosis and induction of some -but not all- NF-κB target genes. The previously reported antioxidant properties of telomerase prompted us to test whether telomerase may be able to modulate ROS-dependent and/or -independent cellular responses to TNF-α, a potent inducer of NF-κB pathway. We showed that telomerase overexpression in normal human fibroblasts, strongly repress activation of ERK1/2 mitogen-activated protein kinases and cell death. In contrast, telomerase overexpression did not affect ROS-independent cellular responses to TNF-α, including NF-κB activation, nuclear translocation of phosphorylated NF-κBp65 and TNF-α-induced transcription of various NF-κB target genes. However, telomerase expression considerably repressed TNF-α-induced transcription of the ROS-sensitive NF-κB target gene SOD2 by reducing ROS contribution to SOD2 gene induction, both in normal fibroblasts and in cancer cells. Our results demonstrate that human telomerase represses ROS-dependent intracellular signaling and gene induction in response to TNF-α without affecting NF-κB activation. Previous studies suggested a possible regulatory role of mouse telomerase on TGF-β signaling pathway. The second aim of this thesis was to investigate whether human telomerase may modulate cellular responses to TGF-β1. We showed that telomerase overexpression in human foreskin fibroblasts does not globally impact on TGF-β signaling as shown by unaltered activation of Smad regulators and similar induction of TGF-β1-target genes. However, telomerase strongly and significantly increased induction of JUNB proto-oncogene transcription in different fibroblast cell lines in response to TGF-β1 treatment. Additional experiments are required to elucidate how human telomerase increase JUNB induction in response to TGF-β1. In summary, our work provides the first evidences for a role of human telomerase in ROS-dependent transcriptional regulation and MAPK activation and further supports the existence of antioxidant functions of telomerase in mitochondria.(SBIM 3) -- UCL, 201

Human telomerase represses ROS-dependent cellular responses to Tumor Necrosis Factor-α without affecting NF-κB activation.

In addition to its well-established role in telomere synthesis, telomerase exerts non-canonical functions that may promote cancer and stem cell survival, notably by lowering reactive oxygen species (ROS) levels and acting as transcriptional cofactor in Wnt-β-catenin signaling pathway. We investigated the impact of telomerase on ROS-dependent and -independent cellular responses to Tumor Necrosis Factor-α (TNF-α), a potent inducer of endogenous ROS production and activator of NF-κB signaling pathway. Strikingly, telomerase overexpression in normal human fibroblasts treated with TNF-α strongly repressed ROS-dependent activation of both ERK1/2 mitogen-activated protein kinases and cell death. Telomerase overexpression also considerably diminished TNF-α-induced transcription of SOD2 Superoxide Dismutase 2 gene by reducing ROS contribution to SOD2 gene induction, both in normal fibroblasts and in cancer cells. Conversely, telomerase did not impair TNF-α-induced transcription of various ROS-insensitive NF-κB target genes. These data were in apparent contrast with the striking observation that telomerase overexpression induced strong constitutive nuclear accumulation of NF-κBp65. Accumulated NF-κBp65, however, lacked Ser-536 activating phosphorylation, was not associated with global constitutive NF-κB activation and did not impair subsequent nuclear translocation of phosphorylated NF-κBp65 in response to TNF-α. Our results demonstrate that human telomerase represses ROS-dependent intracellular signaling and gene induction in response to TNF-α

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-3

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>mm (= 6) and >4.0 mm (= 12) as described in Fig. 1 legend

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-0

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>s calculated as [(cDNA /cDNA ) × 10]. A. expression in normal tissues. Error bars represent standard deviations (SD). The table below the graph gives the average POSTN/ACTB × 10± SD values for each sample. B. expression in primary cultures of human embryonic stem cells at day 5 (HUES), fetal lung fibroblasts (IMR90), newborn foreskin fibroblasts (HFF2) and adult skin fibroblasts (LB2924). Error bars represent SD

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-6

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>s calculated as [(cDNA /cDNA ) × 10]. A. expression in normal tissues. Error bars represent standard deviations (SD). The table below the graph gives the average POSTN/ACTB × 10± SD values for each sample. B. expression in primary cultures of human embryonic stem cells at day 5 (HUES), fetal lung fibroblasts (IMR90), newborn foreskin fibroblasts (HFF2) and adult skin fibroblasts (LB2924). Error bars represent SD

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-5

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>� 10was calculated as [(cDNA /cDNA ) × 10]. Error bars represent SD

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-1

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>nd to samples from Fig. 1. POSTN/ACTB × 10values are given as Mean ± SD in normal tissues (grey bars) and tumors (black bars)

Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells-4

<p><b>Copyright information:</b></p><p>Taken from "Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells"</p><p>http://www.molecular-cancer.com/content/6/1/80</p><p>Molecular Cancer 2007;6():80-80.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222651.</p><p></p>described in Fig. 1 legend. All melanoma cell lines were established in our laboratory and subjected to no more than 4 to 11 passages before RNA extraction. Metastases were isolated from tumor-invaded lymph nodes (LB2201-2, LB2730-1, LB2165-1, LB2117-3, LB2667-1a, LB2800, LB1448-2, LB2870, EB81-2), skin (DDHK0062, KUL81-2), sub-cutaneous tissues (EB124, CP50-1b, LB2174-1, LB2174-3, LB2077-1, LB2821, LB2181-2, LB2763-1, LB2259-1, LB2307-2, VUB105) or heart (LB2077-4). Error bars represent SD. B. expression level was measured in independent melanoma cell lines derived from distinct metastasis of patients LB2174, LB2730 and LB2077. Date of cell line establishment (MM.YY) is indicated above the bars. C. and cDNA levels were measured by qRT-PCR in 12 melanoma cell lines and matched tumors. Values are given as (expression in cell line/expression in tumor) × 100%. D. Periostin protein was analyzed in conditioned medium from LB2077-1 and LB2730-1 periostin-producing melanoma cell lines and from LB2181-2 and LB2667-1a melanoma cell lines, which do not express , as negative controls