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460 Direct Detection of TMPRSS2-ERG Rearrangements in Prostate Cancer by Padlock Probes
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european journal of cancer 48, suppl. 5 (2012) S25–S288
Sunday 8 − Tuesday 10 July 2012
found in 12% of the cases. However a moderate positive correlation (r = 0.646, p < 0.0001) was found between ECADF and ECADC expression. Mean score for Ki67 at the invasive front (KI67F) was 13.9±9.9 and in the tumor core (KI67C) was 9.6±8.2 (p = 0.002). In 72% of the cases Ki67 expression was higher at the invasive front than in the tumor core, and 28% showed Ki67 expression more frequently in the core. There was a regular positive correlation between KI67F and KI67C (r = 0.276, p = 0.036). Although significant, a regular positive correlation between ECADF and KI67C was observed (r = 0.341, p = 0.009), indicating that the E-cadherin loss by tumor cells in EMT at the invasive front did not imply an increase of proliferative status. Conclusion: According to our findings, tumor cell at the invasive front did not show an increased proliferation rates as it loses the E-cadherin expression. A more complex mechanism in the dynamics of E-cadherin and Ki67 expression could be acting during EMT. Further studies are necessary to the better understanding the complexity of this mechanism.
200 levels in primary tumor-derived cell lines. We found miR-200 depletion promoted wound healing and cell migration towards serum. Transplantation of the sponge-expressing lines via tail vein led to in vivo formation of more invasive, metastatic tumors compared to controls. Previously, the miR-200 family has been shown to inhibit the TGF-b target gene and EMT mediator, Zeb1. We have identified another miR-200 target, Epidermal Growth Factor Receptor Substrate 8 (Eps8), which mediates actin based cell motility. In miR200 low (M) cells, Eps8 is expressed three-fold higher than in corresponding miR-200 high (T) cells. Additionally, exogenous expression of Eps8 in miR200 high, T lines promotes wound healing and cell migration, similar to miR200 sponge expressing cells. Thus, our findings implicate the miR-200s as a class of metastasis regulators that modulate expression of genes functionally relevant in tumor cell dissemination and provide new targets for therapeutic intervention of late stage lung adenocarcinoma.
458 Down-modulation of MMP9 Contributes to the Inhibitory Effects of MicroRNA-146a on Melanoma Cell Invasiveness L. Levati1 , E. Pagani1 , P.M. Lacal1 , F. Ruffini1 , E. Bonmassar2 , E. Alvino2 , S. D’Atri1 . 1 IDI-IRCCS, Laboratory of Molecular Oncology, Rome, Italy, 2 National Council of Research, Institute of Translational Pharmacology, Rome, Italy
1 1 S. Kiflemariam1 , M. Mignardi1 , A. Bergh2 , M. Nilsson1 , T. Sjoblom ¨ . Uppsala University /Rudbeck Laboratory, Immunology Genetics and Pathology, ˚ Sweden Uppsala, Sweden, 2 Umea˚ University, Medical Biosciences, Umea,
MicroRNAs (miRNAs), that are post-transcriptional regulators of a large portion of the entire genome, have been implicated in oncogenesis. We previously showed that the majority of melanoma cell lines express miRNA-146a levels lower than those of normal melanocytes. In this study we investigated whether miRNA-146a modulates melanoma cell invasiveness. Two cell lines with normal and two cell lines with low levels of miRNA-146a, respectively, were transfected with with 50 nM of a dsRNA mimicking mature miRNA-146a (miRNA-146a/prec) or of a control dsRNA (dsRNA-CTRL) and analyzed for the ability to invade the extracellular matrix (ECM) 48 h after transfection. ECM invasion was initially evaluated under basal conditions or in response to human fibroblast conditioned medium. The ability to invade the ECM of miRNA-146a/prec-transfected cells was found to be lower (50−70% inhibition) than that of the corresponding dsRNA-CTRL-transfected cells, when the two cell lines under-expressing endogenous miRNA-146a were considered. In contrast, ectopic expression of miRNA-146a did not significantly affect invasiveness of the cell line endowed with normal miRNA-146a levels. Further studies performed in one of the melanoma cell lines with reduced levels of miRNA-146a (i.e WM-115), showed that ECM invasion in response to VEGF-A, but not cell adhesion on ECM, was also markedly impaired (70% inhibition) upon transfection with 50 nM miRNA-146a/prec. To get insight into the possible mechanisms underlying the effects of miRNA-146a on melanoma cell invasiveness, WM-115 cells were transfected with 50 nM miRNA-146a/prec or dsRNA-CTRL and analyzed, by real time RT-PCR, for the expression of a panel of 90 genes involved in cell adhesion, migration and invasion. Several genes resulted differentially expressed in miRNA-146a/prec-transfected cells with respect to control cells. Among the down-regulated genes, MMP9, coding for matrix metalloproteinase-9, was selected for further studies. Downregulation of MMP9 secretion in miRNA-146a/prec-transfected WM-115 cells was confirmed by ELISA assays on cell culture supernatants. Moreover, we found that anti-MMP9 neutralizing antibodies were able to impair VEGF-Ainduced ECM invasion. Our results suggest that down-modulation of MMP9 expression contributes to the inhibitory effects of miRNA-146a on melanoma cell invasiveness. Supported by the Italian Ministry of Health. 459 Loss of the MiR-200s in a Metastatic Model of Lung Adenocarcinoma Promotes Eps8 Mediated Cell Migration and Invasion I.C. Blat1 , M. Winslow2 , T. Jacks1 . 1 Massachusetts Institute of Technology, David H. Koch Institute for Integrative Cancer Research, Cambridge MA, USA, 2 Stanford University, Genetics, Palo Alto CA, USA miRNAs are post-transcriptional regulators of genes involved in such canonical signaling pathways as the TGF-b and EGF pathways, both of which are frequently altered in tumor cells. miRNA downregulation results in upregulation of its target genes and ultimately leads to aberrant signaling of pathways involved in invasion and migration, for example. To better understand how alterations in miRNA expression could lead to metastasis, we used a lung adenocarcinoma mouse model in which introduction of a lentivirus expressing the Cre recombinase leads to expression of oncogenic K-Ras and deletes p53, giving rise to primary lung tumors (T) and metastases (M). We performed comprehensive miRNA expression profiling on 30 T and M derived cell lines and identified three members of the miR-200 family − miR-200b, miR-200c, miR-429 − to be significantly downregulated in the M lines compared to their related T line. To explore the functional contribution of the miR-200s to metastasis, we designed a miRNA sponge to stably inhibit endogenous miR-
460 Direct Detection of TMPRSS2-ERG Rearrangements in Prostate Cancer by Padlock Probes
Background: With the increasing number of translocations known to play a role in the genesis of solid tumors, there is a need for direct tissue localization of gene fusions in molecular pathology. Current methods for detection of TMPRSS2-ERG rearrangements provide qualitative and quantitative information on the rearrangement but no information on the expression of the different fusion variants and their in situ localization. We here present the first technology for multiplex in situ detection of expressed fusion genes. Material and Method: Padlock probes were designed for the most prevalent fusion transcripts (T1/E2, T1/E4, T1/E5, T2/E4 and T2/E5) with the ability to discriminate between them from each other. The wild-type and fusion-positive human prostate cancer cell lines VCaP and LNCaP, respectively, were used to test padlock specificity. After confirming the quality of the probes, the in situ padlock technique was applied on formalin-fixed paraffin-embedded (FFPE) human prostate cancer tissues for identification of expressed TMPRSS2-ERG fusion transcripts. Result and Discussion: We here demonstrate the establishment of multiplex in situ detection of fusion variants in FFPE human prostate cancer tissues by using the prevalent TMPRSS2-ERG translocation. Several TMPRSS2-ERG fusion genes have to date been identified, and we here identify the expression of the most prevalent fusion transcripts (T1/E2, T1/E4, T1/E5, T2/E4 and T2/E5) by using fusion-specific padlock probes and rolling-circle amplification. Conclusion: This novel in situ method holds great potential as a tool to investigate translocations in solid tumors and their role in tumor progression as possible biomarkers or prognostic markers. 461 MEK and SRC Inhibitors as a Combinatorial Approach to Melanoma Therapy J. Ferguson1 , I. Arozarena1 , M. Ehrhardt1 , C. Wellbrock1 . 1 University of Manchester, Manchester, United Kingdom Background: It has been widely shown that the vast majority of malignant melanomas have deregulated signalling in the RAS-RAF-MEK-ERK pathway. These findings have prompted a surge in development of inhibitors targeted to the component kinases of the cascade. In particular, the targeting of MEK is currently being tested in clinical trials. We have looked at the effect of two MEK inhibitors (AZD6244 and PD184352) on melanoma cell-lines in-vitro. As melanoma is a highly metastatic cancer, we have looked closely at the effects of these drugs on adherence and invasion. SRC kinases are known to have a central role in migration and invasion through their regulatory role in focal adhesion formation. A SRC inhibitor (AZD0530) was used in combination with MEK inhibitors to determine whether there was therapeutic potential to this approach. Materials and Method: Experiments were conducted using MEK inhibitors PD184352 and AZD6244, and SRC inhibitor AZD0530. Relative cell invasion through a collagen matrix was determined using an inverted invasion assay and melanoma cell spheres. Relative adhesion was determined by allowing cells to adhere to a collagen substrate for 30−60 min. Gelatine zymography was used to assess MMP activity. MMP expression was shown with qPCR. Proliferation rate was assessed using the Click-iT EdU kit from Invitrogen. Results and Discussion: SRC inhibition had little impact on cells entering S-phase in melanoma cell-lines but striking anti-invasive effects. This was characterised by a significant loss in adherence to collagen substrate. However, we have found that whilst MEK inhibition can efficiently suppress tumour cell growth, it can also increase cell invasiveness. After treatment with MEK inhibitor, cells displayed an increase in integrin-mediated adhesion
european journal of cancer 48, suppl. 5 (2012) S25–S288
Sunday 8 − Tuesday 10 July 2012
found in 12% of the cases. However a moderate positive correlation (r = 0.646, p < 0.0001) was found between ECADF and ECADC expression. Mean score for Ki67 at the invasive front (KI67F) was 13.9±9.9 and in the tumor core (KI67C) was 9.6±8.2 (p = 0.002). In 72% of the cases Ki67 expression was higher at the invasive front than in the tumor core, and 28% showed Ki67 expression more frequently in the core. There was a regular positive correlation between KI67F and KI67C (r = 0.276, p = 0.036). Although significant, a regular positive correlation between ECADF and KI67C was observed (r = 0.341, p = 0.009), indicating that the E-cadherin loss by tumor cells in EMT at the invasive front did not imply an increase of proliferative status. Conclusion: According to our findings, tumor cell at the invasive front did not show an increased proliferation rates as it loses the E-cadherin expression. A more complex mechanism in the dynamics of E-cadherin and Ki67 expression could be acting during EMT. Further studies are necessary to the better understanding the complexity of this mechanism.
200 levels in primary tumor-derived cell lines. We found miR-200 depletion promoted wound healing and cell migration towards serum. Transplantation of the sponge-expressing lines via tail vein led to in vivo formation of more invasive, metastatic tumors compared to controls. Previously, the miR-200 family has been shown to inhibit the TGF-b target gene and EMT mediator, Zeb1. We have identified another miR-200 target, Epidermal Growth Factor Receptor Substrate 8 (Eps8), which mediates actin based cell motility. In miR200 low (M) cells, Eps8 is expressed three-fold higher than in corresponding miR-200 high (T) cells. Additionally, exogenous expression of Eps8 in miR200 high, T lines promotes wound healing and cell migration, similar to miR200 sponge expressing cells. Thus, our findings implicate the miR-200s as a class of metastasis regulators that modulate expression of genes functionally relevant in tumor cell dissemination and provide new targets for therapeutic intervention of late stage lung adenocarcinoma.
458 Down-modulation of MMP9 Contributes to the Inhibitory Effects of MicroRNA-146a on Melanoma Cell Invasiveness L. Levati1 , E. Pagani1 , P.M. Lacal1 , F. Ruffini1 , E. Bonmassar2 , E. Alvino2 , S. D’Atri1 . 1 IDI-IRCCS, Laboratory of Molecular Oncology, Rome, Italy, 2 National Council of Research, Institute of Translational Pharmacology, Rome, Italy
1 1 S. Kiflemariam1 , M. Mignardi1 , A. Bergh2 , M. Nilsson1 , T. Sjoblom ¨ . Uppsala University /Rudbeck Laboratory, Immunology Genetics and Pathology, ˚ Sweden Uppsala, Sweden, 2 Umea˚ University, Medical Biosciences, Umea,
MicroRNAs (miRNAs), that are post-transcriptional regulators of a large portion of the entire genome, have been implicated in oncogenesis. We previously showed that the majority of melanoma cell lines express miRNA-146a levels lower than those of normal melanocytes. In this study we investigated whether miRNA-146a modulates melanoma cell invasiveness. Two cell lines with normal and two cell lines with low levels of miRNA-146a, respectively, were transfected with with 50 nM of a dsRNA mimicking mature miRNA-146a (miRNA-146a/prec) or of a control dsRNA (dsRNA-CTRL) and analyzed for the ability to invade the extracellular matrix (ECM) 48 h after transfection. ECM invasion was initially evaluated under basal conditions or in response to human fibroblast conditioned medium. The ability to invade the ECM of miRNA-146a/prec-transfected cells was found to be lower (50−70% inhibition) than that of the corresponding dsRNA-CTRL-transfected cells, when the two cell lines under-expressing endogenous miRNA-146a were considered. In contrast, ectopic expression of miRNA-146a did not significantly affect invasiveness of the cell line endowed with normal miRNA-146a levels. Further studies performed in one of the melanoma cell lines with reduced levels of miRNA-146a (i.e WM-115), showed that ECM invasion in response to VEGF-A, but not cell adhesion on ECM, was also markedly impaired (70% inhibition) upon transfection with 50 nM miRNA-146a/prec. To get insight into the possible mechanisms underlying the effects of miRNA-146a on melanoma cell invasiveness, WM-115 cells were transfected with 50 nM miRNA-146a/prec or dsRNA-CTRL and analyzed, by real time RT-PCR, for the expression of a panel of 90 genes involved in cell adhesion, migration and invasion. Several genes resulted differentially expressed in miRNA-146a/prec-transfected cells with respect to control cells. Among the down-regulated genes, MMP9, coding for matrix metalloproteinase-9, was selected for further studies. Downregulation of MMP9 secretion in miRNA-146a/prec-transfected WM-115 cells was confirmed by ELISA assays on cell culture supernatants. Moreover, we found that anti-MMP9 neutralizing antibodies were able to impair VEGF-Ainduced ECM invasion. Our results suggest that down-modulation of MMP9 expression contributes to the inhibitory effects of miRNA-146a on melanoma cell invasiveness. Supported by the Italian Ministry of Health. 459 Loss of the MiR-200s in a Metastatic Model of Lung Adenocarcinoma Promotes Eps8 Mediated Cell Migration and Invasion I.C. Blat1 , M. Winslow2 , T. Jacks1 . 1 Massachusetts Institute of Technology, David H. Koch Institute for Integrative Cancer Research, Cambridge MA, USA, 2 Stanford University, Genetics, Palo Alto CA, USA miRNAs are post-transcriptional regulators of genes involved in such canonical signaling pathways as the TGF-b and EGF pathways, both of which are frequently altered in tumor cells. miRNA downregulation results in upregulation of its target genes and ultimately leads to aberrant signaling of pathways involved in invasion and migration, for example. To better understand how alterations in miRNA expression could lead to metastasis, we used a lung adenocarcinoma mouse model in which introduction of a lentivirus expressing the Cre recombinase leads to expression of oncogenic K-Ras and deletes p53, giving rise to primary lung tumors (T) and metastases (M). We performed comprehensive miRNA expression profiling on 30 T and M derived cell lines and identified three members of the miR-200 family − miR-200b, miR-200c, miR-429 − to be significantly downregulated in the M lines compared to their related T line. To explore the functional contribution of the miR-200s to metastasis, we designed a miRNA sponge to stably inhibit endogenous miR-
460 Direct Detection of TMPRSS2-ERG Rearrangements in Prostate Cancer by Padlock Probes
Background: With the increasing number of translocations known to play a role in the genesis of solid tumors, there is a need for direct tissue localization of gene fusions in molecular pathology. Current methods for detection of TMPRSS2-ERG rearrangements provide qualitative and quantitative information on the rearrangement but no information on the expression of the different fusion variants and their in situ localization. We here present the first technology for multiplex in situ detection of expressed fusion genes. Material and Method: Padlock probes were designed for the most prevalent fusion transcripts (T1/E2, T1/E4, T1/E5, T2/E4 and T2/E5) with the ability to discriminate between them from each other. The wild-type and fusion-positive human prostate cancer cell lines VCaP and LNCaP, respectively, were used to test padlock specificity. After confirming the quality of the probes, the in situ padlock technique was applied on formalin-fixed paraffin-embedded (FFPE) human prostate cancer tissues for identification of expressed TMPRSS2-ERG fusion transcripts. Result and Discussion: We here demonstrate the establishment of multiplex in situ detection of fusion variants in FFPE human prostate cancer tissues by using the prevalent TMPRSS2-ERG translocation. Several TMPRSS2-ERG fusion genes have to date been identified, and we here identify the expression of the most prevalent fusion transcripts (T1/E2, T1/E4, T1/E5, T2/E4 and T2/E5) by using fusion-specific padlock probes and rolling-circle amplification. Conclusion: This novel in situ method holds great potential as a tool to investigate translocations in solid tumors and their role in tumor progression as possible biomarkers or prognostic markers. 461 MEK and SRC Inhibitors as a Combinatorial Approach to Melanoma Therapy J. Ferguson1 , I. Arozarena1 , M. Ehrhardt1 , C. Wellbrock1 . 1 University of Manchester, Manchester, United Kingdom Background: It has been widely shown that the vast majority of malignant melanomas have deregulated signalling in the RAS-RAF-MEK-ERK pathway. These findings have prompted a surge in development of inhibitors targeted to the component kinases of the cascade. In particular, the targeting of MEK is currently being tested in clinical trials. We have looked at the effect of two MEK inhibitors (AZD6244 and PD184352) on melanoma cell-lines in-vitro. As melanoma is a highly metastatic cancer, we have looked closely at the effects of these drugs on adherence and invasion. SRC kinases are known to have a central role in migration and invasion through their regulatory role in focal adhesion formation. A SRC inhibitor (AZD0530) was used in combination with MEK inhibitors to determine whether there was therapeutic potential to this approach. Materials and Method: Experiments were conducted using MEK inhibitors PD184352 and AZD6244, and SRC inhibitor AZD0530. Relative cell invasion through a collagen matrix was determined using an inverted invasion assay and melanoma cell spheres. Relative adhesion was determined by allowing cells to adhere to a collagen substrate for 30−60 min. Gelatine zymography was used to assess MMP activity. MMP expression was shown with qPCR. Proliferation rate was assessed using the Click-iT EdU kit from Invitrogen. Results and Discussion: SRC inhibition had little impact on cells entering S-phase in melanoma cell-lines but striking anti-invasive effects. This was characterised by a significant loss in adherence to collagen substrate. However, we have found that whilst MEK inhibition can efficiently suppress tumour cell growth, it can also increase cell invasiveness. After treatment with MEK inhibitor, cells displayed an increase in integrin-mediated adhesion