Nicole Grabinski

Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells

BackgroundDue to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients.MethodsThe activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells.ResultsAKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient.ConclusionOur results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.

Distinct functional roles of Akt isoforms for proliferation, survival, migration and EGF-mediated signalling in lung cancer derived disseminated tumor cells.

Single disseminated tumor cells (DTC) can be detected in the bone marrow (BM) from 20% to 60% of patients with various tumors including non-small cell lung cancer (NSCLC). Detection of DTC in the BM of NSCLC patients is associated with poor prognosis and may be responsible for metastatic relapse. However, the functional properties of DTC are widely unknown. Here, we performed the first functional analysis of DTC focusing on the activation of the PI3K/Akt signalling pathway and the functional roles of Akt isoforms. In vitro kinase assays revealed a high activity of Akt3 in NSCLC-derived DTC. Proliferation and survival of DTC was reduced by depletion of Akt3 and to a lesser extend by Akt1, but not after depletion of Akt2. The major effect of Akt3 on the proliferation of DTC was associated with an Akt3-mediated regulation of both, cyclin D1 and cyclin D3, whereas Akt1 regulated the expression of cyclin D1 only. In contrast all three Akt isoforms, especially Akt2, were involved in the regulation of migration. Analysis of signalling events downstream of distinct Akt isoforms revealed that expression levels of urokinase-type plasminogen activator and its receptor were decreased after knockdown of Akt1 and Akt3. In addition, EGF-stimulated proliferative and anti-apoptotic signals are mediated by Akt1 and Akt3 in DTC. Finally, by immunofluorescence staining of primary DTC from BM samples of lung cancer patients, pAkt(S473) and Akt3 positive DTC were detected in vivo. Our data demonstrate that Akt1 and notably Akt3 regulate proliferation, survival, migration and EGF-mediated signal transduction in NSCLC-derived DTC.

Combined targeting of AKT and mTOR using MK-2206 and RAD001 is synergistic in the treatment of cholangiocarcinoma

Cholangiocarcinoma (CCA) is a rare, but devastating disease arising from the epithelium of intrahepatic and extrahepatic bile ducts. There are neither effective systemic therapies nor satisfying treatment options for inoperable CCA. Histopathological and biochemical studies of CCA show frequent dysregulation of the phosphatidylinositol 3‐kinase/AKT/mammalian target of rapamycin (mTOR) pathway. Therefore, we investigated the efficacy of the mTOR inhibitor RAD001 and the impact of AKT signaling following mTOR inhibition in the treatment of CCA. RAD001 significantly inhibits proliferation of CCA cell lines, however, a concentration‐dependent and isoform specific feedback activation of the three AKT isoforms (AKT1, AKT2 and AKT3) was observed after mTOR inhibition. As activation of AKT might limit the RAD001‐mediated anti‐tumor effect, the efficacy of combined mTOR and AKT inhibition was investigated using the allosteric AKT inhibitor MK‐2206. Our results show that inhibition of AKT potentiates the efficacy of mTOR inhibition both in vitro and in a xenograft mouse model in vivo. Mechanistically, the antiproliferative effect of the pan‐AKT inhibitor MK2206 in the CCA cell line TFK‐1 was due to inhibition of AKT1 and AKT2, because knockdown of either AKT1 or AKT2, but not AKT3, showed a synergistic reduction of cell proliferation in combination with mTOR treatment. Finally, using an AKT isoform specific in vitro kinase assay, enzymatic activity of each of the three AKT isoforms was detected in all tissue samples from CCA patients, analyzed. In summary, our preclinical data suggest that combined targeting of mTOR and AKT using RAD001 and MK‐2206 might be a new, effective strategy for the treatment of CCA.

AKT3 regulates ErbB2, ErbB3 and estrogen receptor α expression and contributes to endocrine therapy resistance of ErbB2(+) breast tumor cells from Balb-neuT mice.

ErbB2(+) breast cancer is an aggressive breast cancer subtype generally associated with lower estrogen receptor alpha (ERα) expression and more aggressive tumor behavior compared to ERα(+)/ErbB2(-) breast cancer. The ErbB2(+) phenotype is associated with resistance to endocrine therapy, e.g. the selective estrogen receptor modulator Tamoxifen. However, the mechanisms underlying endocrine resistance are not fully understood. Here, we investigated the impact of AKT signaling and distinct functional roles of AKT isoforms in ErbB2(+) breast cancer from Balb-neuT mice. AKT isoform specific in vitro kinase assays revealed that AKT3 is activated in Balb-neuT breast tumors in comparison to normal murine breast tissue. Knock-down of AKT3, but not of AKT1 or AKT2, led to reduced expression and tyrosine-phosphorylation of ErbB2 and ErbB3 in Balb-neuT-derived mammary tumor cells. In contrast, expression of ERα was strongly up-regulated and phosphorylation of the AKT substrate Foxo3a which regulates ERα transcription was decreased in AKT3 knockdown cells. These data suggest that ERα expression is down regulated via AKT3/Foxo3a signaling in ErbB2(+) breast cancer cells. Furthermore, up-regulation of ERα after depletion of AKT3 resulted in a significant increase in Tamoxifen responsiveness of Balb-neuT-derived mammary tumor cells. In addition, Tamoxifen resistant human breast cancer cell lines showed increased AKT3 expression and activity in comparison to Tamoxifen responsive MCF-7 cells. Finally, by AKT isoform specific in vitro kinase assays of human breast cancer samples, AKT3 activity was detected in ErbB2(+) and triple negative tumors but not in ERα(+) breast cancer. Our data indicate that AKT3 regulates the expression of ErbB2, ErbB3 and ERα and demonstrate that down-regulation of activated AKT3 can sensitize ErbB2(+) breast cancer cells for treatment with Tamoxifen. Therefore, AKT3 targeting might be a new promising strategy for therapy of ErbB2(+)/ERα(-) breast cancer and might further increase the responsiveness to an endocrine therapy approach.

ErbB2 signaling activates the Hedgehog pathway via PI3K–Akt in human esophageal adenocarcinoma: Identification of novel targets for concerted therapy concepts

The Hedgehog pathway plays an important role in the pathogenesis of several tumor types, including esophageal cancer. In our study, we show an expression of the ligand Indian hedgehog (Ihh) and its downstream mediator Gli-1 in primary resected adenocarcinoma tissue by immunohistochemistry and quantitative PCR in fifty percent of the cases, while matching healthy esophagus mucosa was negative for both proteins. Moreover, a functionally important regulation of Gli-1 by ErbB2-PI3K-mTORC signaling as well as a Gli-1-dependent regulation of Ihh in the ErbB2 amplified esophageal adenocarcinoma cell line OE19 was observed. Treatment of OE19 cells with the Her2 antibody trastuzumab, the PI3K-mTORC1 inhibitor NVP BEZ235 (BEZ235) or the knockdown of Akt1 resulted in a downregulation of Gli-1 and Ihh as well as in a reduction of viable OE19 cells in vitro. Interestingly, the Hedgehog receptor Smo, which acts upstream of Gli-1, was not expressed in OE19 cells and in the majority of primary human esophageal adenocarcinoma, suggesting a non-canonical upregulation of Gli-1 expression by the ErbB2-PI3K axis. To translate our findings into a therapeutic concept, we targeted ErbB2-PI3K-mTORC1 by trastuzumab and BEZ235, combining both compounds with the Gli-1/2 inhibitor GANT61. The triple combination led to significantly stronger reduction of tumor cell viability than cisplatinum or each biological alone. Therefore, concomitant blockage of the ErbB2-PI3K pathway and the Hedgehog downstream mediator Gli-1 may provide a new therapeutic strategy for esophageal cancer.

Plasminogen activator inhibitor type 1 up-regulation is associated with skeletal muscle atrophy and associated fibrosis.

Muscle wasting remains a feature of many diseases and is counteracted by anabolic supplementation or exercise. Persisting atrophy-inducing conditions can be complicated by skeletal muscle fibrosis, which leads to functional impairment. Identification of early mechanisms that initiate atrophy-induced fibrosis may reveal novel targets for therapy or diagnosis. Therefore, we investigated changes in the expression of genes involved in extracellular matrix homeostasis during glucocorticoid-induced atrophy of myotubes and compared them with insulin-like growth factor-1-induced hypertrophy. Obtained results were verified in rat gastrocnemius muscle that was exposed to microgravity by space flight for 2 weeks. Myostatin and atrogin-1 mRNA levels reflected the magnitude of atrophy. Despite differential induction of these negative muscle mass regulators, no major changes in matrix metalloproteinases-2, -9, and -14 mRNAs or their physiological inhibitors could be detected in either atrophy model. In contrast, transcript levels of plasminogen activator inhibitor type 1 (PAI-1) was dramatically increased in atrophic myotubes and microgravity-exposed rat gastrocnemius muscle, while plasminogen activators remained unaltered. In contrast to atrophy, no increase in PAI-1 mRNA levels could be detected in rat hindlimb that was electrically stimulated for 21 days. Furthermore, a strong increase in PAI-1 mRNA levels was identified in skeletal muscle of patients with neurogenic muscle atrophy. Our study suggests that increased PAI-1 expression in atrophic skeletal muscle may lead to muscle fibrosis by reducing plasmin generation.

The inositol 5-phosphatase SHIP1 is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei.

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in hematopoietic cells. SHIP1 mediates its regulatory function after relocalization from the cytoplasm to the plasma membrane where it converts its substrate PI(3,4,5)P(3) to PI(3,4)P(2) thereby terminating PI3-kinase mediated signaling. In addition, SHIP1 converts Ins(1,3,4,5)P(4) to Ins(1,3,4)P(3) thereby regulating inositol phosphate metabolism. Here we report, that SHIP1 can be detected in nuclear puncta of Jurkat cells by confocal microscopy after expression of SHIP1 from a tetracycline inducible vector. SHIP1-containing nuclear puncta partially co-localize with FLASH, a multifunctional nuclear protein that has been linked to apoptotic signaling and transcriptional control. Nuclear localization was confirmed for endogenously expressed SHIP1 in the myeloid leukemia cell line TF1. In addition, enzymatically active SHIP1 was found in nuclear fractions of Jurkat cells with a similar specific activity as cytoplasmic SHIP1. Further analysis revealed that SHIP1 is a nucleocytoplasmic shuttling protein which is actively imported into and exported out of the nucleus. Nuclear import is mediated by two canonical nuclear localization signals (NLS) i.e. K(327)KSK and K(547)KLR. Mutational inactivation of each NLS motif inhibited nuclear import and reduced the proliferation of cells indicating a functional role of nuclear SHIP1 for cell growth. Our data indicate that SHIP1 is partly localized in the nucleus and suggest that SHIP1 plays a role for nuclear phosphoinositide and/or nuclear inositol phosphate signaling.

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo. Methods The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo. Results Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3. Conclusions We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.

Abstract 4830: Precision cut cancer tissues slices as human model for the testing of immuno-modulatory compounds

The goal of personalized medicine is to stratify individual patients to the appropriate treatment. This approach depends on extensive characterization of individual tumors and their sensitivity to therapeutics. In the context of the immunotherapy of cancer, information on the localization, abundance and activation of immune cells within individual tumors gained in importance. In this study, we showed that viable tumors from colorectal cancer patients used within our drug testing platform, exhibit different populations of infiltrating immune cells. Analysis of immune cells was conducted on disaggregated, cells from viable tumor slices. Disaggregation of precision cut cancer tissue slices was performed using the GentleMACS from Miltenyi. Immune cell subsets were analyzed by flow cytometric multiplexing of CD3, CD4, CD8 and CD45. Furthermore, we identified PD1 positive cells among the CD45+/CD3+ lymphocyte population, indicating relevance for anti-PD1 targeted therapy in colorectal cancer. As a proof of principle, precision cut cancer tissue slices were incubated for 24 hours with different concentrations of Nivolumab (anti-PD1). The read out of treatment effects was conducted in regard to cytokine secretions upon compound treatment as well as immune cell composition. Cytokines were analyzed in supernatants of tissue cultures using the proinflammatory panel from Mesoscale Discovery. The results demonstrated that immune cell compositions were stable and uniform within our precision cut cancer tissue slices both pre- and post-cultivation, and pre- and post-treatment with Nivolumab. In contrast, cytokine secretion had changed after treatment. This has been observed for different cytokines, such as INF gamma, IL-2, IL-10 and TNF alpha. A correlation to PD-1 expression on T-cells in different patients has not been seen. In order to optimize preclinical testing of immune-modulatory compounds, preclinical models, which reflect the individual tumor, as well as the individual immune components of the tumor, are mandatory. We have shown here that effects of treatments with an immune-modulatory compound (Nivolumab) were detectable in this system. Therefore, this drug testing platform represents a unique opportunity to test immune-modulatory compounds in a fully human, patient derived model that is close to in vivo situation. In the future, other immune-modulatory classes of compounds have to be tested within the system to more comprehensively elucidate the possibilities and limits of this drug testing platform in regard to immunotherapy. Citation Format: Kristina Bernoth, Florian T. Unger, Moiken Petersen, Mirja Piller, Jana Kruger, Nicole Grabinski, Hartmut Juhl, Kerstin A. David. Precision cut cancer tissues slices as human model for the testing of immuno-modulatory compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4830. doi:10.1158/1538-7445.AM2017-4830