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  • 1
    Online Resource
    Online Resource
    Abstract 803: A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism
    American Association for Cancer Research (AACR) ; 2014
    In:  Cancer Research Vol. 74, No. 19_Supplement ( 2014-10-01), p. 803-803
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 74, No. 19_Supplement ( 2014-10-01), p. 803-803
    Abstract: Preclinical evidence shows that hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loop, may cause paradoxical pathway activation and may lead to functional resistance. Thus, the “vertical” combination of agents simultaneously inhibiting RAF and MEK has been proposed as a strategy to synergistically inhibit tumor growth and delay resistance. Experimental procedures: Molecular and functional effects of single and combined MEK (using trametinib, T), BRAF (using dabrafenib, D), and RAF (using the pan-RAF inhibitor, RAF265, R) inhibition were dissected by WB analysis and conservative isobologram analysis to assess functional synergism, using a fixed dose-ratio experimental design. Summary data: We examined the in vitro molecular and functional consequences of D and T, alone or in combination, in a panel of different human BRAFV600E melanoma cell lines; both drugs inhibited cell growth and inactivated the MAPK pathway, but little or no synergistic growth inhibition was observed with their combination (CI: 0.7 - 1.3x106). Conversely, combined D+T suppressed malignant growth with highly synergistic effects in KRAS-mutant lung (2/5 cell lines tested) and pancreatic (4/6 cell lines tested) cancer models (CI: 0.1 - 0.7), in which selective BRAF inhibition induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect). At concentrations inhibiting both BRAF and CRAF, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. To define the role of RAS gene status in determining sensitivity/resistance to single and combined RAF and MEK blockade, we analyzed two isogenic tumor cell line models: H1299 expressing individual codon 12 KRAS mutants and isogenic HCT116 clones differing for the presence of a homo or heterozygous G13D KRAS mutation. Conversely, in lung cancer models driven by either EGFR mutations (HCC827, H1650) or HER-2 overexpression (Calu-3), selective BRAF inhibition also induced a paradox MAPK activation, which could be blocked using a reversible EGFR/HER-2 inhibitor (Lapatinib); in this context, combination (D+T) resulted in a non-synergic growth inhibitory effects. Conclusions: Overall, our data indicate that, in appropriate cellular contexts, vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects across different cancer models. Citation Format: Anais Del Curatolo, Ursula Cesta Incani, Ludovica Ciuffreda, Italia Falcone, Senji Shirasawa, Massimo Broggini, Isabella Sperduti, Adriana Eramo, Ruggero De Maria, Francesco Cognetti, Michele Milella. A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2014-803
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2014-803
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2014
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    detail.hit.zdb_id: 410466-3
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  • 2
    Online Resource
    Online Resource
    Loss of FGFR4 promotes the malignant phenotype of PDAC
    Springer Science and Business Media LLC ; 2022
    In:  Oncogene Vol. 41, No. 38 ( 2022-09-16), p. 4371-4384
    Details
    In: Oncogene, Springer Science and Business Media LLC, Vol. 41, No. 38 ( 2022-09-16), p. 4371-4384
    Abstract: Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.
    Type of Medium: Online Resource
    ISSN: 0950-9232 , 1476-5594
    URL: Article
    DOI: 10.1038/s41388-022-02432-5
    RVK:
    XA 10000
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
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  • 3
    Online Resource
    Online Resource
    Synergistic activity of vertical combinations of agents targeting multiple steps along the RAF/MEK/ERK cascade as a therapeutic strategy in human tumors.
    American Society of Clinical Oncology (ASCO) ; 2012
    In:  Journal of Clinical Oncology Vol. 30, No. 15_suppl ( 2012-05-20), p. e13572-e13572
    Details
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 30, No. 15_suppl ( 2012-05-20), p. e13572-e13572
    Abstract: e13572 Background: BRAF-selective kinase inhibitors have potent antitumor effects in mutant BRAF(V600E) tumors; however, in BRAF-wt cells, they paradoxically activate MEK/ERK. In addition, MEK blockade may induce compensatory signaling through both upstream pathway elements (RAF) and parallel pathways (PI3K/AKT/mTOR). Methods: We set out to define molecular and functional effects of single and combined BRAF (GSK2118436A, BRAF-I) and MEK (GSK1120212B, MEK-I) inhibition, using WB analysis to dissect signaling and a fixed dose-ratio experimental design to assess functional synergism by conservative isobologram analysis. Results: In A549 lung adenocarcinoma (KRAS G12S), BRAF-I (10 μM) induces hyperphosphorylation of CRAF, MEK, ERK, and p90RSK, while MEK-I (10 nM), alone or in combination with BRAF-I, potently offsets MAPK activation. Combined BRAF-I and MEK-I suppress malignant growth and survival at 72 h with highly synergistic effects in the A549 (lung, KRAS G12S), H1299 (lung, NRAS), HCT116 (colon, KRAS G13D), and MIAPACA (pancreatic, KRAS G12V) models, with combination indexes (CI), ranging from 1.37 to 0.12. Conversely, in other lung cancer models (H460, Calu-1, Calu-3) the combination of BRAF-I and MEK-I produced modestly additive to highly antagonistic antitumor effects. In BRAF-mutant melanoma and colon carcinoma models (M14 and HT29), there was no paradoxical activation of the MEK/ERK module in response to BRAF-I and both BRAF-I and MEK-I had pronounced growth inhibitory effects as single agents, but were frankly antagonistic in combination. Similarly, the pan-RAF inhibitor RAF265 did not cause MAPK activation and did not result in synergistic growth inhibition when combined with the MEK-I in the A549 and MIAPACA cell lines. Conclusions: Overall, our data indicate that combined inhibition of multiple signaling elements along the RAF/MEK/ERK pathway results in strongly synergistic growth inhibition, particularly in tumors with RAS mutations. Additional studies to better define genetic determinants of sensitivity/resistance and molecular mechanisms of therapeutic synergism of combined BRAF-I and MEK-I are currently ongoing.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    URL: Article
    DOI: 10.1200/jco.2012.30.15_suppl.e13572
    RVK:
    XA 52760
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2012
    detail.hit.zdb_id: 2005181-5
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  • 4
    Online Resource
    Online Resource
    Abstract 2618: PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade
    American Association for Cancer Research (AACR) ; 2014
    In:  Cancer Research Vol. 74, No. 19_Supplement ( 2014-10-01), p. 2618-2618
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 74, No. 19_Supplement ( 2014-10-01), p. 2618-2618
    Abstract: Background. Complex feedback loops and crosstalk between the MEK/ERK and PI3K/mTOR pathways, thus molecular predictors of sensitivity/resistance and synergistic/antagonistic interactions are urgently needed for the effective clinical development of combination strategies. Here we hypothesize that PTEN status critically modulates the growth inhibitory activity of single and combined MEK and PI3K/mTOR inhibition. Methods. Molecular and functional effects of single and combined MEK (trametinib, T) and mTOR (everolimus, E) blockade were assessed in a panel of 29 cancer cell lines with different molecular ‘drivers’. Pharmacologic interactions were analyzed by conservative isobologram analysis. PTEN role was mechanistically assessed by either silencing PTEN expression by shRNA or overexpressing a functional PTEN protein by stable transfection. Specific proteins and their phosphorylation states were analyzed using Kinexus Antibody Microarrays. Results. In the cell line panel analyzed, the presence of a wt-PTEN was the only significant predictor of sensitivity to T (p=0.001), while BRAF mutations were significantly related to E resistance (p=0.015). PTEN silencing slightly increased resistance to T in the wt-PTEN melanoma cell line M14; conversely, wt-PTEN overexpression in the PTEN-del WM115 cell line, dramatically increased sensitivity to T. E-induced growth inhibition was not significantly affected. Combined MEK and mTOR inhibition resulted in a striking growth inhibitory synergism in cells lacking PTEN expression (CI: 0.4-0.0005), but not in those with an intact PTEN (CI: 1.2-107; p=0.001). However, PTEN silencing restored growth inhibitory synergism of combined T and E in M14 cells (CI:0.36); similarly, the slope of the CI/fraction affected curve was dramatically altered in PTEN-overexpressing WM115 cells, as compared to their control-transfected counterpart, again indicating that PTEN expression/function causally influences functional response to combined MEK and mTOR inhibition. Similar results in terms of synergistic/antagonistic pharmacologic interactions were obtained when either double PI3K/mTOR kinase inhibitors (PF-5212384) or AKT allosteric inhibitors (MK-2206) were used to block the PI3K/mTOR pathway in combination with T. Proteomic analysis indicated that a greater modification of protein expression/phosphorylation profiles in response to MEK or combined MEK/mTOR inhibition occurs in cells lacking a functional PTEN, as compared to the wt-PTEN-expressing ones; preliminary analysis suggests that AKT phosphorylation and NF-κB activation maybe be crucial mediators of synergistic growth-inhibitory interactions occurring with combined treatment. Conclusions. PTEN loss may constitute a suitable genetic/molecular marker of synergistic activity interactions between MEK/ERK and PI3K/mTOR pathway inhibitors. Citation Format: Ludovica Ciuffreda, Italia Falcone, Silvia Matteoni, Andrea Sacconi, Federico Malusa, Teresa De Luca, Ursula Cesta Incani, Anais Del Curatolo, Marina Konopleva, Michael Andreeff, Adriana Eramo, Ruggero De Maria, Donatella Del Bufalo, Francesco Cognetti, Michele Milella. PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2014-2618
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2014-2618
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2014
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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  • 5
    Online Resource
    Online Resource
    Use of PTEN status to determine the functional outcome of combined MEK and mTOR inhibition.
    American Society of Clinical Oncology (ASCO) ; 2012
    In:  Journal of Clinical Oncology Vol. 30, No. 15_suppl ( 2012-05-20), p. e13512-e13512
    Details
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 30, No. 15_suppl ( 2012-05-20), p. e13512-e13512
    Abstract: e13512 Background: PTEN is a lipid phosphatase couteracting the activity of the PI3K pathway. PTEN mutations and deficiencies are prevalent in many types of human cancers and are associated with poor prognosis and therapeutic resistance. MAPK is another key cellular network that works independently from, in parallel to, and/or through interconnections with PI3K to promote cancer development. Here we investigated whether a strategy combining MEK and mTOR inhibition may be effective in preclinical models of human cancer and the role of PTEN loss in determining sensivity/resistance to single and combined pathway inhibition. Methods: We employed in vitro assays (cell proliferation assays, cell cycle and apoptosis analysis, WB, and ELISA assays) to determine functional and molecular drug effects. Pharmacologic interactions between MEK and mTOR inhibitors were analyzed by conservative isobologram analysis using a fixed dose-ratio experimental design. Results: In cell lines with wt-PTEN (including melanoma, breast, lung, and colon cancer), combined MEK and mTOR blockade achieves synergistic effects at suboptimal drug concentrations but becomes frankly antagonistic in the presence of complete inhibition of MEK-to-ERK signaling (combination index - CI: 1.2- 〉 1000). This, in turn, led to the identification of a novel crosstalk mechanism by which MEK blockade restores PTEN expression and cross-inhibits the PI3K/AKT/mTOR pathway, thus bypassing the need for double pathway blockade. Conversely, in cancer cell lines with PTEN loss, combined MEK and mTOR blockade resulted in strongly synergistic effects (CI: 0.0005-0.4), due to cooperative induction of apoptosis. A similar synergistic growth inhibition was also observed in a patient-derived lung cancer stem cell line, which displayed low to undetectable PTEN protein levels. Conclusions: Our results suggest that combined MEK and mTOR inhibition exerts strongly synergistic effects in cancer models with PTEN loss. This notion may be helpful in selecting appropriate cellular (and possibly clinical) contexts for the design rational therapeutic strategies based on concomitant inhibition of the RAF/MEK/ERK and PI3K/AKT/mTOR pathways.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    URL: Article
    DOI: 10.1200/jco.2012.30.15_suppl.e13512
    RVK:
    XA 52760
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2012
    detail.hit.zdb_id: 2005181-5
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