Project description:Senescent cells secrete a plethora of factors with potent paracrine signaling capacity. Strikingly, senescence, which acts as a defense against cell transformation, exerts pro-tumorigenic activities through its secretome by promoting numerous tumor-specific features, such as cellular proliferation, epithelial-mesenchymal transition and invasiveness. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the unique activity of activating cell death exclusively in tumor cells. Given that the senescence-associated secretome supports cell transformation, we asked whether factor(s) of this secretome would establish a program required for the acquisition of TRAIL sensitivity. We found that conditioned media from several types of senescent cells (CMS) efficiently sensitized pre-transformed cells to TRAIL, while the same was not observed with normal or immortalized cells. Dynamic transcription profiling analysis of CMS-exposed pre-transformed cells revealed paracrine autoregulatory loop of senescence-associated secretome factors and a dominant role of CMS-induced MYC. Sensitization to TRAIL coincided with MYC upregulation and massive changes in gene regulation. CMS-induced MYC silenced its target gene CFLAR, encoding the apoptosis inhibitor FLIPL, thus leading to the acquisition of TRAIL sensitivity. Altogether, our results reveal that senescent cell-secreted factors exert a TRAIL sensitizing effect on pre-transformed cells by modulating the expression of MYC and CFLAR. Notably, CMS dose-dependent sensitization to TRAIL was observed with TRAIL-insensitive cancer cells and confirmed in co-culture experiments. Dissection and characterization of TRAIL-sensitizing CMS factors and the associated signaling pathway(s) may provide a mechanistic insight in the acquisition of TRAIL sensitivity and lead to novel concepts for the apoptogenic therapy of pre-malignant and TRAIL-resistant tumors. Pre-transformed BJEL cells were incubated with CMS for 0, 1, 3, 6, 8, 16 or 24 h respectively. Total RNA has been extracted from each time point and used for gene expression analysis (Affymetrix Human Gene 1.0 ST Arrays).

Project description:Melanocytes within benign human nevi are the paradigm for tumor suppressive senescent cells in a pre-malignant neoplasm. These cells typically contain mutations in either the BRAF or N-RAS oncogene and express markers of senescence, including p16. However, a nevus can contain 10s to 100s of thousands of clonal melanocytes and approximately 20-30% of melanoma are thought to arise in association with a pre-existing nevus. Neither observation is indicative of fail-safe senescence-associated proliferation arrest and tumor suppression. We set out to better understand the status of nevus melanocytes. Proliferation-promoting Wnt target genes, such as cyclin D1 and c-myc, were repressed in oncogene-induced senescent melanocytes in vitro, and repression of Wnt signaling in these cells induced a senescent-like state. In contrast, cyclin D1 and c-myc were expressed in many melanocytes of human benign nevi. Specifically, activated Wnt signalling in nevi correlated inversely with nevus maturation, an established dermatopathological correlate of clinical benignancy. Single cell analyses of lone epidermal melanocytes and nevus melanocytes showed that expression of proliferation-promoting Wnt targets correlates with prior proliferative expansion of p16-expressing nevus melanocytes. In a mouse model, activation of Wnt signaling delayed, but did not bypass, senescence of oncogene-expressing melanocytes, leading to massive accumulation of proliferation-arrested, p16-positive non-malignant melanocytes. We conclude that clonal hyperproliferation of oncogene-expressing melanocytes to form a nevus is facilitated by transient delay of senescence due to activated Wnt signaling. The observation that activation of Wnt signaling correlates inversely with nevus maturation, an indicator of clinical benignancy, supports the notion that persistent destabilization of senescence by Wnt signaling contributes to the malignant potential of nevi. We used RNA-Seq to detail the global programme of gene expression in human melanoma cell lines

Project description:Melanocytes within benign human nevi are the paradigm for tumor suppressive senescent cells in a pre-malignant neoplasm. These cells typically contain mutations in either the BRAF or N-RAS oncogene and express markers of senescence, including p16. However, a nevus can contain 10s to 100s of thousands of clonal melanocytes and approximately 20-30% of melanoma are thought to arise in association with a pre-existing nevus. Neither observation is indicative of fail-safe senescence-associated proliferation arrest and tumor suppression. We set out to better understand the status of nevus melanocytes. Proliferation-promoting Wnt target genes, such as cyclin D1 and c-myc, were repressed in oncogene-induced senescent melanocytes in vitro, and repression of Wnt signaling in these cells induced a senescent-like state. In contrast, cyclin D1 and c-myc were expressed in many melanocytes of human benign nevi. Specifically, activated Wnt signalling in nevi correlated inversely with nevus maturation, an established dermatopathological correlate of clinical benignancy. Single cell analyses of lone epidermal melanocytes and nevus melanocytes showed that expression of proliferation-promoting Wnt targets correlates with prior proliferative expansion of p16-expressing nevus melanocytes. In a mouse model, activation of Wnt signaling delayed, but did not bypass, senescence of oncogene-expressing melanocytes, leading to massive accumulation of proliferation-arrested, p16-positive non-malignant melanocytes. We conclude that clonal hyperproliferation of oncogene-expressing melanocytes to form a nevus is facilitated by transient delay of senescence due to activated Wnt signaling. The observation that activation of Wnt signaling correlates inversely with nevus maturation, an indicator of clinical benignancy, supports the notion that persistent destabilization of senescence by Wnt signaling contributes to the malignant potential of nevi. We used RNA-Seq to detail the global programme of gene expression in primary human melanocytes which were Uninfected and BRAF600V induced cells

Project description:Melanocytes within benign human nevi are the paradigm for tumor suppressive senescent cells in a pre-malignant neoplasm. These cells typically contain mutations in either the BRAF or N-RAS oncogene and express markers of senescence, including p16. However, a nevus can contain 10s to 100s of thousands of clonal melanocytes and approximately 20-30% of melanoma are thought to arise in association with a pre-existing nevus. Neither observation is indicative of fail-safe senescence-associated proliferation arrest and tumor suppression. We set out to better understand the status of nevus melanocytes. Proliferation-promoting Wnt target genes, such as cyclin D1 and c-myc, were repressed in oncogene-induced senescent melanocytes in vitro, and repression of Wnt signaling in these cells induced a senescent-like state. In contrast, cyclin D1 and c-myc were expressed in many melanocytes of human benign nevi. Specifically, activated Wnt signalling in nevi correlated inversely with nevus maturation, an established dermatopathological correlate of clinical benignancy. Single cell analyses of lone epidermal melanocytes and nevus melanocytes showed that expression of proliferation-promoting Wnt targets correlates with prior proliferative expansion of p16-expressing nevus melanocytes. In a mouse model, activation of Wnt signaling delayed, but did not bypass, senescence of oncogene-expressing melanocytes, leading to massive accumulation of proliferation-arrested, p16-positive non-malignant melanocytes. We conclude that clonal hyperproliferation of oncogene-expressing melanocytes to form a nevus is facilitated by transient delay of senescence due to activated Wnt signaling. The observation that activation of Wnt signaling correlates inversely with nevus maturation, an indicator of clinical benignancy, supports the notion that persistent destabilization of senescence by Wnt signaling contributes to the malignant potential of nevi. We used microarrays to detail the global programme of gene expression after lentiviral infection of BRAF in 3 replicates. Primary human melanocytes were infected/uninfected with lentivirus containing either an Empty Vector or activated BRAFV600E mutant in three biological replicates

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Senescence secreted factors activate Myc and sensitize pre-transformed cells to TRAIL-induced apoptosis

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