- Email: [email protected]
Sos1 disruption increases mitochondrial oxidative stress in primary MEFs
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 a European collection of HCC and to gain information regarding the molecular regulation of this system in hepatocellular cells. Material and Methods: We used 85 paired tumour/adjacent nontumour (NT) liver samples, normal human hepatocytes in primary culture and four human HCC cell lines. Immunohistochemistry (IHC), real time RT-PCR, ELISA, Western blot and cell migration assays were performed. Cell lines were treated either with cycloheximide, a protein synthesis inhibitor, MG132, a proteasome inhibitor or Akt inhibitor VIII. mRNA expression was downregulated using smallinterferent RNA (siRNA). Results: HER3 mRNA was increased in HCC versus paired NT tissue while HRG-1b mRNA was lower in HCC (p < 0.0001; median fold-variation: 1.94 and 0.09, respectively). Expression ratios T/NT for HER3 and HRG-1b were not associated with histological markers reminiscent of tumours with bad outcome. Spearman correlation analysis indicated no correlation between HER3 mRNA and protein levels in HCC and NT samples (Spearman r=-0.230; p = 0.071). HER3 analysis by IHC revealed predominant cytoplasmic staining but was nonreproducible in terms of intensity. In vitro experiments showed that HER3 was associated to insulin receptor (IR) in HCC cell lines. In HepG2 and PLC/PRF5 cell lines as well as in normal hepatocytes, insulin induced the proteasomal degradation of HER3 protein through an AKT-dependent pathway. This effect was not observed in Hep3B and HuH7 cells but in these cell lines insulin inhibited HRG-1b stimulation of cell migration. In addition, the downregulation of IR expression with siRNA markedly enhanced the stimulatory effect of HRG-1b. Conclusion: Although overexpression of HER3 mRNA was observed in our HCC collection, it was not associated with poor prognosis markers. The discrepancy between the expression level of transcript and protein suggests that HER3 is subjected to post-transcriptional regulation in liver tissue. We identified insulin signaling as a negative regulator of the expression and function of HER3. No conflict of interest. 739 BRAF and Src/FAK are required for TGFb-induced EMT of thyroid cancer cells 2 ´ , A. Santos2 , B. Gallego2 , M. Lasa3 , P. Baquero1 , E. Jimenez-Mora A. Chiloeches2 . 1 University of Glasgow, Wolfson Wohl Cancer Research Centre- College of Medical- Veterinary & Life Sciences- Institute of Cancer ´ System Sciences, Glasgow, United Kingdom, 2 Universidad de Alcala, Biology- Unit Biochemistry and Molecular Biology, Alcala´ de Henares- Madrid, 3 ´ de Madrid- Instituto de Investigaciones Spain, Universidad Autonoma ´ Biomedicas “Alberto Sols”- CSIC-UAM, Bioqu´ımica, Madrid, Spain The epithelial–mesenchymal transition (EMT) is a crucial process in tumour progression, by which epithelial cells acquire a mesenchymal phenotype, increasing its motility and the ability to invade distant sites. Here, we describe the molecular mechanisms by which V600EBRAF, TGFb and the Src/FAK complex cooperatively regulate EMT induction and cell motility of anaplastic thyroid cancer cells. Analysis of EMT marker levels reveals a positive correlation between TGFb and Snail expression, with a concomitant downregulation of E-cadherin, accompanied by an increase of cell migration and invasion. Furthermore, we show that V600EBRAF depletion by siRNA or inhibition of its activity by treatment with its inhibitor PLX4720 reverses the TGFb-mediated effects on Snail, E-cadherin, migration and invasion. In addition, TGFb activates the Src/FAK complex, which in turn regulates the expression of Snail and E-cadherin as well as cell migration. The inhibition of Src with the inhibitor SU6656 or abrogation of FAK expression with a specific siRNA reverses the TGFb-induced effects. Interestingly, we demonstrate that activation of the Src/FAK complex by TGFb is independent of V600EBRAF signalling, since inhibition of this oncogene does not affect its phosphorylation. Our data strongly suggest that TGFb induces EMT and aggressiveness of thyroid cancer cells by parallel mechanisms involving both the BRAF/MEK/ERK and Src/FAK pathways independently. Thus, we describe novel functions for Src/FAK in mediating the EMT program and aggressiveness regulated by TGFb, establishing the inhibition of these proteins as a possible effective approach in preventing tumour progression of V600EBRAF-expressing thyroid tumours. No conflict of interest. 740 Sos1 disruption increases mitochondrial oxidative stress in primary MEFs 1 ´ , R. Garc´ıa-Navas1 , P. Liceras-Boillos1 , B. Anta1 , C. Lillo2 , C. Gomez D. Jimeno1 , N. Calzada1 , M.S. Jimenez1 , F.C. Baltanas ´ 1 , E. Santos1 . 1 ´ Instituto de Biolog´ıa Celular y Molecular del Cancer, Laboratory 01, Salamanca, Spain, 2 Instituto de Neurociencias de Castilla y Leon, Laboratory 3, Salamanca, Spain
The Sos family of RasGEF activators encompasses two highly homologous members (Sos1 and Sos2) which are ubiquitously expressed and appear to function in multiple signaling pathways, but their specific functional properties have not yet been clearly defined. Using a tamoxifen (4OHT)-inducible,
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conditional Sos1 null mutation, we analyzed wild type (WT), single Sos1 KO, Sos2 KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEFs) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1 KO and Sos1/2 DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2 KO counterparts. The Sos1 KO and Sos1/2 DKO MEFs also displayed significant accumulation, in comparison to WT and Sos2 KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1 KO and the Sos1/2 DKO cells as compared to Sos2 KO and WT MEFs. Treatment of the MEF cultures with cell-scavengers-antioxidants like GSH and NAC corrected the altered phenotypes of Sos1 KO and Sos1/2 DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2 KO MEFs. Interestingly, treatment with the specific mitochondrial superoxide scavenger mitoTEMPO recovered endogenous redox-homeostasis in Sos1 KO and Sos1/2 DKO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability. Acknowledgement: This work was supported by AECC, grants FS/24-2015, PI13/02846 (FIS) and RD12/0036/0001(RTICC) from ISCIII. No conflict of interest. 741 The dual inhibition of RNA Pol I transcription and PIM kinase as a new therapeutic approach to treat advanced prostate cancer R. Rebello1 , E. Kusnadi2 , D. Cameron2 , H. Pearson2 , A. Lesmana2 , J. Devlin2 , D. Drygin3 , A. Clark1 , L. Porter1 , J. Pedersen4 , S. Sandhu5 , G. Risbridger1 , R. Pearson2 , R. Hannan6 , L. Furic1 . 1 Biomedicine Discovery Institute- Monash University, Cancer Program- Anatomy & Developmental Biology, Clayton, Australia, 2 Peter MacCallum Cancer Centre, Oncogenic Signalling and Growth Control Program, East Melbourne, Australia, 3 PimeraInc., Pimera, San Diego, USA, 4 TissuPath Pathology, TissuPath, Melbourne, Australia, 5 Peter MacCallum Cancer Centre, Sir Peter MacCallum Department of Oncology, Parkville, Australia, 6 Australian National University, Department of Cancer Biology and Therapeutics, ACT, Australia Background: The MYC oncogene is commonly over-expressed in prostate cancer (PC). Upregulation of ribosome biogenesis and function is a characteristic feature of MYC-driven tumours. Accordingly, inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription has been successfully exploited therapeutically in models of hematological malignancy characterized by elevated MYC expression. Additionally, PIM kinases activate MYC signaling and mRNA translation in PC and cooperate with MYC to accelerate tumorigenesis. Here, we investigate the efficacy of a dual approach targeting ribosome biogenesis and function to treat PC by combining CX-5461 with the pan-PIM kinase inhibitor CX-6258. Materials and Methods: The efficacy of CX-5461 and CX-6258, alone and in combination, was tested in PC cell lines, in the Hi-MYC mouse model and in PC metastatic tissue obtained from castration-resistant PC patient. Results: CX-5461 reduced anchorage-independent growth and induced cell cycle arrest in human PC cell lines and in primary prostatic epithelial cells from Hi-MYC mice. CX-5461 treatment of Hi-MYC mice induced p53 expression and activity and significantly reduced prostate epithelial cell proliferation and invasion. While CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation, dramatically reduced the incidence of large invasive lesions and reverted lesions to prostatic intraepithelial neoplasia. This promising combination strategy prevented the growth of therapy-resistant, patient-derived xenograft tissue characterized by elevated MYC. Conclusions: Our results demonstrate the preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of PC with high MYC activity. No conflict of interest. 742 ETV5 as a mediator of the oncogenic effects of mutant FGFR3 in bladder cancer E. Di Martino1 , M. Knowles1 . 1 University of Leeds, Experimental Oncology, Leeds, United Kingdom Background: Alteration of fibroblast growth factor receptor 3 (FGFR3) signalling is a common event in bladder cancer and targeted therapies are in clinical trial. Identification of key effectors and downstream targets of mutant FGFR3 will aid patient selection and development of second line treatments. By comparing gene expression profiles in normal urothelial cells
The Sos family of RasGEF activators encompasses two highly homologous members (Sos1 and Sos2) which are ubiquitously expressed and appear to function in multiple signaling pathways, but their specific functional properties have not yet been clearly defined. Using a tamoxifen (4OHT)-inducible,
S167
conditional Sos1 null mutation, we analyzed wild type (WT), single Sos1 KO, Sos2 KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEFs) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1 KO and Sos1/2 DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2 KO counterparts. The Sos1 KO and Sos1/2 DKO MEFs also displayed significant accumulation, in comparison to WT and Sos2 KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1 KO and the Sos1/2 DKO cells as compared to Sos2 KO and WT MEFs. Treatment of the MEF cultures with cell-scavengers-antioxidants like GSH and NAC corrected the altered phenotypes of Sos1 KO and Sos1/2 DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2 KO MEFs. Interestingly, treatment with the specific mitochondrial superoxide scavenger mitoTEMPO recovered endogenous redox-homeostasis in Sos1 KO and Sos1/2 DKO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability. Acknowledgement: This work was supported by AECC, grants FS/24-2015, PI13/02846 (FIS) and RD12/0036/0001(RTICC) from ISCIII. No conflict of interest. 741 The dual inhibition of RNA Pol I transcription and PIM kinase as a new therapeutic approach to treat advanced prostate cancer R. Rebello1 , E. Kusnadi2 , D. Cameron2 , H. Pearson2 , A. Lesmana2 , J. Devlin2 , D. Drygin3 , A. Clark1 , L. Porter1 , J. Pedersen4 , S. Sandhu5 , G. Risbridger1 , R. Pearson2 , R. Hannan6 , L. Furic1 . 1 Biomedicine Discovery Institute- Monash University, Cancer Program- Anatomy & Developmental Biology, Clayton, Australia, 2 Peter MacCallum Cancer Centre, Oncogenic Signalling and Growth Control Program, East Melbourne, Australia, 3 PimeraInc., Pimera, San Diego, USA, 4 TissuPath Pathology, TissuPath, Melbourne, Australia, 5 Peter MacCallum Cancer Centre, Sir Peter MacCallum Department of Oncology, Parkville, Australia, 6 Australian National University, Department of Cancer Biology and Therapeutics, ACT, Australia Background: The MYC oncogene is commonly over-expressed in prostate cancer (PC). Upregulation of ribosome biogenesis and function is a characteristic feature of MYC-driven tumours. Accordingly, inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription has been successfully exploited therapeutically in models of hematological malignancy characterized by elevated MYC expression. Additionally, PIM kinases activate MYC signaling and mRNA translation in PC and cooperate with MYC to accelerate tumorigenesis. Here, we investigate the efficacy of a dual approach targeting ribosome biogenesis and function to treat PC by combining CX-5461 with the pan-PIM kinase inhibitor CX-6258. Materials and Methods: The efficacy of CX-5461 and CX-6258, alone and in combination, was tested in PC cell lines, in the Hi-MYC mouse model and in PC metastatic tissue obtained from castration-resistant PC patient. Results: CX-5461 reduced anchorage-independent growth and induced cell cycle arrest in human PC cell lines and in primary prostatic epithelial cells from Hi-MYC mice. CX-5461 treatment of Hi-MYC mice induced p53 expression and activity and significantly reduced prostate epithelial cell proliferation and invasion. While CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation, dramatically reduced the incidence of large invasive lesions and reverted lesions to prostatic intraepithelial neoplasia. This promising combination strategy prevented the growth of therapy-resistant, patient-derived xenograft tissue characterized by elevated MYC. Conclusions: Our results demonstrate the preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of PC with high MYC activity. No conflict of interest. 742 ETV5 as a mediator of the oncogenic effects of mutant FGFR3 in bladder cancer E. Di Martino1 , M. Knowles1 . 1 University of Leeds, Experimental Oncology, Leeds, United Kingdom Background: Alteration of fibroblast growth factor receptor 3 (FGFR3) signalling is a common event in bladder cancer and targeted therapies are in clinical trial. Identification of key effectors and downstream targets of mutant FGFR3 will aid patient selection and development of second line treatments. By comparing gene expression profiles in normal urothelial cells