Vertical Targeting of AKT and mTOR as Well as Dual Targeting of AKT and MEK Signaling Is Synergistic in Hepatocellular Carcinoma

Ewald, Florian; Nörz, Dominik; Grottke, Astrid; Bach, Johanna; Herzberger, Christiane; Hofmann, Bianca T.; Nashan, Björn; Jücker, Manfred

doi:10.7150/jca.12452

Cited by 37 publications

(32 citation statements)

References 61 publications

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“…Firstly, RAF1 is found downregulated in a non-selected cohort of human HCC samples; secondly, modelling RAF1 downregulation in human HCC cells, in culture or in xenografts, increases cell proliferation; and thirdly, the same results are obtained in two independent genetic models (AlfpCre and MxCre-induced RAF1 ablation in the DEN/PB-treated mice) and in premalignant hepatocytes derived from these models. This consistency is remarkable given the molecular heterogeneity of human HCC as well as of the human cell lines studied30 and the transgenic models used. The finding was entirely unexpected as the existing literature unanimously points to pro-tumourigenic functions of RAF1.…”

Section: Discussionmentioning

confidence: 82%

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

et al. 2016

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Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, but its molecular heterogeneity hampers the design of targeted therapies. Currently, the only therapeutic option for advanced HCC is Sorafenib, an inhibitor whose targets include RAF. Unexpectedly, RAF1 expression is reduced in human HCC samples. Modelling RAF1 downregulation by RNAi increases the proliferation of human HCC lines in xenografts and in culture; furthermore, RAF1 ablation promotes chemical hepatocarcinogenesis and the proliferation of cultured (pre)malignant mouse hepatocytes. The phenotypes depend on increased YAP1 expression and STAT3 activation, observed in cultured RAF1-deficient cells, in HCC xenografts, and in autochthonous liver tumours. Thus RAF1, although essential for the development of skin and lung tumours, is a negative regulator of hepatocarcinogenesis. This unexpected finding highlights the contribution of the cellular/tissue environment in determining the function of a protein, and underscores the importance of understanding the molecular context of a disease to inform therapy design.

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Section: Discussionmentioning

confidence: 82%

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

et al. 2016

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“…17,34 Dual blockade of the mTOR pathway has been shown to be synergistic for other cancer types, such as hepatocellular carcinoma. 35,36 In addition, dual blockade can overcome certain resistances that arise in cancer cell lines, as demonstrated in neuroblastoma cells. 37 Therefore, the observed synergy of dual PI3K/Akt/mTOR blockade in our PTLD model has precedence in other cancer types, and is a rational strategy for overcoming the modest efficacy of rapamycin monotherapy in PTLD patients.…”

Section: Treatment Of Ebv-associated Ptld Remains a Clinical Challengmentioning

confidence: 99%

Dual blockade of the PI3K/Akt/mTOR pathway inhibits posttransplant Epstein-Barr virus B cell lymphomas and promotes allograft survival

Sang

McPherson

Ivison

et al. 2019

American Journal of Transplantation

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Abbreviations: 4EBP, 4E-binding protein; DMSO, dimethyl sulfoxide; EBV, Epstein-Barr virus; ERK, extracellular signal-regulated kinase; IC 50 , half maximal Inhibitory concentration; LI, Loewe index; LMP1, latent membrane protein 1; MAPK, mitogen-activated protein kinase; mTORC, mammalian target of rapamycin complex; mTOR, mammalian target of rapamycin; NOD-SCID, Non-obese diabetic/severe combined immunodeficiency; PI3K, Phospho-inositide 3-kinase; PTLD, posttransplant lymphoproliferative disorder; RI, reduction of immunosuppression; SEM, standard error of the mean; STAT, signal transducer and activator of transcription. Posttransplant lymphoproliferative disorder (PTLD) is a serious complication of organ transplantation that often manifests as Epstein-Barr virus (EBV)-associated B cell lymphomas. Current treatments for PTLD have limited efficacy and can be associated with graft rejection or systemic toxicities. The mTOR inhibitor, rapamycin, suppresses tumor growth of EBV+ B cell lymphoma cells in vitro and in vivo; however, the efficacy is limited and clinical benefits of mTOR inhibitors for PTLD are variable. Here, we show constitutive activation of multiple nodes within the PI3K/Akt/mTOR pathway in EBV+ PTLD-derived cell lines. Inhibition of either PI3K or Akt, with specific inhibitors CAL-101 and MK-2206, respectively, diminished growth of EBV+ B cell lines from PTLD patients in a dose-dependent manner. Importantly, rapamycin combined with CAL-101 or MK-2206 had a synergistic effect in suppressing cell growth as determined by IC 50 isobolographic analysis and Loewe indices. Moreover, these combinations were significantly more effective than rapamycin alone in inhibiting tumor xenograft growth in NOD-SCID mice. Finally, both CAL-101 and MK-2206 also prolonged survival of heterotopic cardiac allografts in C57BL/6 mice. Thus, combination therapy with rapamycin and a PI3K inhibitor, or an Akt inhibitor, can be an efficacious treatment for EBV-associated PTLD, while simultaneously promoting allograft survival. K E Y W O R D S basic (laboratory) research/science, immunosuppressant-mechanistic target of rapamycin (mTOR), immunosuppression/immune modulation, infection and infectious agents-viral: Epstein-Barr Virus (EBV), infectious disease, posttransplant lymphoproliferative disorder (PTLD)

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“…For inhibitor treatment in 2D and 3D cultures, 100 nM of rapamycin (Grabiner et al, 2014;Kang et al, 2013;Sarbassov et al, 2006), 250 nM of Torin1 (Cheng et al, 2016;Grabiner et al, 2014;Kang et al, 2013;Thoreen et al, 2012), 10 μM of PF47068671 (Pearce et al, 2010;Schipany et al, 2015;Zhang et al, 2015), 1 μM of MK2206 (Devery et al, 2015;Shen et al, 2015;Sung et al, 2016) and 1 μM of AZD6244 (Devery et al, 2015;Ewald et al, 2015) were employed as these concentrations have been most frequently used in cell-based assays and have a complete effect on their respective targets [ phospho-protein analysis in western blots (see Fig. 4 and western blots in the supporting figures)].…”

Section: Inhibitor Treatmentmentioning

confidence: 99%

Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response

Riedl

Schlederer

Pudelko

et al. 2016

Journal of Cell Science

349

293

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Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo. These 3D models displayed augmented antitumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.

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Vertical Targeting of AKT and mTOR as Well as Dual Targeting of AKT and MEK Signaling Is Synergistic in Hepatocellular Carcinoma

Cited by 37 publications

References 61 publications

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

Dual blockade of the PI3K/Akt/mTOR pathway inhibits posttransplant Epstein-Barr virus B cell lymphomas and promotes allograft survival

Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response

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