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

Abstract Background Due 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. Methods The 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. Results AKT 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. Conclusion Our 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.</p

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

Hypocretin/orexin increases the expression of steroidogenic enzymes in human adrenocortical NCI H295R cells

Wenzel J, Grabinski N, Knopp CA, Dendorfer A, Ramanjaneya M, Randeva HS, Ehrhart-Bornstein M, Dominiak P, Johren O. Hypocretin/orexin increases the expression of steroidogenic enzymes in human adrenocortical NCI H295R cells. Am J Physiol Regul Integr Comp Physiol 297: R1601-R1609, 2009. First published September 30, 2009; doi: 10.1152/ajpregu. 91034.2008.-Hypocretins/orexins act through two receptor subtypes: OX1 and OX2. Outside the brain, orexin receptors are expressed in adrenal glands, where orexins stimulate the release of glucocorticoids. To further address the regulation of steroidogenesis, we analyzed the effect of orexins on the expression of steroidogenic enzymes in human adrenocortical National Cancer Institute (NCI) H295R cells by qPCR. In NCI H295R cells, OX2 receptors were highly expressed, as they were in human adrenal glands. After treatment of NCI H295R cells with orexin A for 12-24 h, the cortisol synthesis rate was significantly increased, whereas 30 min of treatment showed no effect. While CYP11B1 and CYP11B2 mRNA levels were increased already at earlier time points, the expression of HSD3B2 and CYP21 mRNA was significantly up-regulated after treatment with orexin A for 12 h. Likewise, orexin B increased CYP21 and HSD3B2 mRNA levels showing, however, a lower potency compared with orexin A. The mRNA levels of CYP11A and CYP17 were unaffected by orexin A. OX2 receptor mRNA levels were down-regulated after 12 and 24 h of orexin A treatment. Orexin A increased intracellular Ca2+ but not cAMP concentrations in NCI H295R cells. Furthermore, inhibition of PKC and MAPK kinase/ERK kinase (MEK1/2) prevented the increase of HSD3B2 expression by orexin A. Accordingly, orexin A treatment of NCI H295R cells markedly enhanced ERK1/2 phosphorylation that was prevented by PKC and, in part, PKA inhibition. In conclusion, orexins may influence adrenal steroidogenesis by differential regulation of the expression of steroidogenic enzymes involving Ca2+, as well as PKC-ERK1/2 signaling

S100A4 is overexpressed in AKT3 lacking primary tumors.

<p>(A) Protein lysates were prepared from five primary tumors per group and expression of S100A4 was analyzed by Western blot analysis. (B) S100A4 expression was quantified from Western blot analysis of five primary tumor tissue samples. Data were normalized to corresponding HSC70 controls (Bars: SD. **, p < 0,01, ***, p < 0,001). (C) Immunohistochemical staining of S100A4 in xenograft tumor sections. One representative image is shown (magnification 200X).</p

Knockdown of AKT3 increases the metastatic potential of MDA-MB-231 cells <i>in vivo</i>.

<p>AKT2,3 and AKT1,3 double knockdown cells as well as control cells were injected subcutaneously into SCID mice (8 to 10 mice per group). Tumors were allowed to grow until a termination criterion was met. (A) Growth period and tumor weight upon necropsy (Bars: SD, ns., p>0,05). (B) The number of disseminated tumor cells in the left lung of mice was quantified by Alu-PCR. (Bars: SD, *, p<0,05) (C) Protein lysates were prepared from 5 tumors per group, and AKT isoform knockdown were analyzed by Western blot. (D) Representative images from histological examination of the right lungs from mice injected with AKT2,3 and AKT1,3 knockdown cells showing macroscopically detectable metastases.</p

Knockdown of AKT3 increases the expression of S100A4 protein.

<p>(A) AKT signaling pathway activity after downregulation of AKT isoforms was analyzed by Western blot with antibodies directed against the indicated proteins. HSC70 was used as loading control. (B) Relative mRNA levels of S100A4 were measured by qPCR (Bars: SD. *, p < 0,05. **, p < 0,01). (C) Expression of S100A4 in MDA-MB-231 control and AKT isoform knockdown cells was analyzed by Western blot, HSC70 was used as loading control. (D) S100A4 expression was quantified from Western blot analysis of triplicates and data were normalized to corresponding HSC70 controls (Bars: SD. *, p < 0,05).</p

Depletion of AKT3 increases transmigration and chemotaxis in MDA-MB-231 cells.

<p>(A) For transwell assays, cells were seeded into the upper chamber of transwell filters and allowed to migrate towards a FCS gradient for 16 h. Cells that have reached the lower side of the transwell insert were stained with Calcein AM, and fluorescence signal was detected using a Tecan Infinite 200M reader (Bars: SD. **, p < 0,01). (B) For chemotaxis analysis, cells were seeded into the observation area of chemotaxis slides, and reservoirs were filled to generate a stable FCS gradient (final concentration of 10%). Time lapse images were recorded to analyze the effective chemotaxis (Bars: SD. *, p < 0,05).</p

Effect of AKT isoform single and double knockdown on migration and proliferation.

<p>(A) Knockdowns of AKT isoforms in MDA-MB-231 cells were performed by lentiviral transduction using AKT isoform specific shRNAs. Efficiency was confirmed by Western blot analysis. (B)-(C) Analysis of cell migration using scratch assay technique. A confluent monolayer was scratched using a 200μl pipette tip and cell migration was analyzed using time lapse video microscopy. Mean single cell velocity (B) of MDA-MB-231 control and AKT isoform knockdown cells. One experiment out of three is shown (Bars: SD. *, p < 0,05, **, p < 0,01. ***, p < 0,001). (C) Representative image of a scratch assay after 0 and 24 hours, the dotted line indicates the leading front. (D) Proliferation was analyzed by manual cell counting over four consecutive days, performed in triplicates, proliferation is shown as relative cell count compared to day 0 (Bars: SD, n.s., p>0,05).</p

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