Impact of AKT1 on cell invasion and radiosensitivity in a triple negative breast cancer cell line developing brain metastasis.

Introduction: The PI3K/AKT pathway is activated in 43-70% of breast cancer (BC)-patients and promotes the metastatic potential of BC cells by increasing cell proliferation, invasion and radioresistance. Therefore, AKT1-inhibition in combination with radiotherapy might be an effective treatment option for triple-negative breast cancer (TNBC)-patients with brain metastases. Methods: The impact of AKT1-knockout (AKT1_KO) and AKT-inhibition using Ipatasertib on MDA-MB-231 BR cells was assessed using in vitro cell proliferation and migration assays. AKT1-knockout in MDA-MB-231BR cells was performed using CRISPR/Cas9. The effect of AKT1-knockout on radiosensitivity of MDA-MB-231BR cell lines was determined via colony formation assays after cell irradiation. To detect genomic variants in AKT1_KO MDA-MB-231BR cells, whole-genome sequencing (WGS) was performed. Results: Pharmacological inhibition of AKT with the pan-AKT inhibitor Ipatasertib led to a significant reduction of cell viability but did not impact cell migration. Moreover, only MDA-MB-231BR cells were sensitized following Ipatasertib-treatment. Furthermore, specific AKT1-knockout in MDA-MB-231BR showed reduced cell viability in comparison to control cells, with significant effect in one of two analyzed clones. Unexpectedly, AKT1 knockout led to increased cell migration and clonogenic potential in both AKT1_KO clones. RNAseq-analysis revealed the deregulation of CTSO, CYBB, GPR68, CEBPA, ID1, ID4, METTL15, PBX1 and PTGFRN leading to the increased cell migration, higher clonogenic survival and decreased radiosensitivity as a consequence of the AKT1 knockout in MDA-MB-231BR. Discussion: Collectively, our results demonstrate that Ipatasertib leads to radiosensitization and reduced cell proliferation of MDA-MB-231BR. AKT1-inhibition showed altered gene expression profile leading to modified cell migration, clonogenic survival and radioresistance in MDA-MB-231BR. We conclude, that AKT1-inhibition in combination with radiotherapy contribute to novel treatment strategies for breast cancer brain metastases.

Investigation of the function of the PI3-Kinase / AKT signaling pathway for leukemogenesis and therapy of acute childhood lymphoblastic leukemia (ALL).

Acute lymphoblastic leukemia is the most common cause of cancer-related death in children and, especially for patients in a high-risk group, still represents a poor prognosis. The PI3K/AKT/mTOR signaling pathway has been identified as a frequently constitutively activated switching point in the disease of ALL. Despite the knowledge of the therapeutic importance of the signaling pathway, the results of clinically effective treatment strategies have so far been extremely sobering. In particular, monotherapy approaches represent a major problem with regard to cell resistance. In this work, the PI3K/AKT/mTOR signaling pathway was examined as a therapeutic target for the treatment of childhood acute lymphoblastic leukemia (ALL) with a new therapeutic approach to avoid cell resistance. Therefore, we used a combined therapeutic approach with inhibitors directed against AKT (MK2206), mTOR (RAD001) and the most prominent and aberrantly activated tyrosine kinase. In case of BCR-ABL-positive B-ALL cells we used a combination with the classic inhibitor Imatinib and in case of MLL-AF4-positive B-ALL cells we used a combination with Quizartinib (directed against FLT3). We show, in particular compared to the monotherapies, a highly significant inhibition of the growth of these cells after this new specific triple combination therapy. Furthermore, we show that inhibiting AKT alone leads to a feedback mechanism and an upregulation of the phosphorylation of a number of receptor-tyrosine-kinases. After isoform-specific knockdown of the three AKT isoforms in ALL cells we identified that especially ErbB2/Her2 is most strongly phosphorylated in cells with AKT2 knockdown. AKT isoform 1 and 2 knockdown cells show, in contrast to AKT isoform 3 knockdown cells, a weak proliferation and are presumably kept alive among others by the increased phosphorylation of the receptor-tyrosine-kinase ErbB2. This work provides first indications for a new combination therapy of B-ALL cells, which is directed against AKT, mTOR and a predominantly highly activated kinase.

Cerebral metastasis in recurrent squamous cell carcinoma of the vulva: case report and review of the literature.

BACKGROUND: Distant metastases from squamous cell cancer of the vulva (VSCC) are encountered rarely and are associated with a poor prognosis. Cerebral metastases have only been described anecdotally. CASE HISTORY: A 51-year old woman was diagnosed with hepatic metastases due to VSCC. Initial therapy comprised wide local excision of the primary tumor with inguino-femoral lymphadenectomy (LAE) followed by stereotactic radiation of the singular hepatic metastasis while adjuvant chemoradiation of the vulva and lymphatics was declined. 3 years later, she subsequently developed lung and cerebral metastases. CONCLUSION: The course of metastatic disease in VSCC is poorly understood. Further knowledge of the metastatic patterns in vulvar cancer is required for guidance of future therapeutic interventions.

Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells.

Akt1 through the C-terminal domain interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and stimulates the repair of DNA double-strand breaks (DSBs) in K-RAS-mutated (K-RASmut) cells. We investigated the interactions of distinct domain(s) of DNA-PKcs in binding to full-length Akt1. Similarly, we analyzed potential interactions of DNA-PKcs with Akt2 and Akt3. Finally the effect of Akt isoforms in cell proliferation and tumor growth was tested. We demonstrated that Akt1 preferentially binds to the N-terminal domain of DNA-PKcs using pull-down studies with distinct eGFP-tagged DNA-PKcs fragments that were expressed by plasmids in combination with mCherry-tagged full-length Akt isoforms. These binding studies also indicated an interaction with the intermediate and C-terminal domains of DNA-PKcs. In contrast, Akt3 interacted with all four DNA-PKcs fragments without a marked preference for any specific domain. Notably, we could not see binding of Akt2 to any of the tested DNA-PKcs fragments. In subsequent studies, we demonstrated that Akt inhibition interferes with binding of Akt1 to the N-terminal domain of DNA-PKcs. This indicated a correlation between Akt1 activity and the Akt1/DNA-PKcs complex formation. Finally, knockdown studies revealed that the depletion of endogenous Akt1 and Akt3, but not Akt2, inhibit clonogenic activity and repair of ionizing radiation (IR)-induced DNA DSBs, leading to radiosensitization. Furthermore, in a xenograft study the expression of shAkt1 or shAkt3, but not shAkt2 in K-RASmut breast cancer cell line MDA-MB-231 showed major tumor growth delay. Together, these data indicate that Akt1 and Akt3, but not Akt2, physically interact with DNA-PKcs, thus stimulating the repair of DSBs and therefore protecting K-RASmut cells against IR. Likewise, interaction of Akt isoforms with DNA-PKcs could be crucial for their role in regulating tumor growth.

PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression.

Despite improvement of therapeutic treatments for breast cancer, the development of brain metastases has become a major limitation to life expectancy for many patients. Brain metastases show very commonly alterations in EGFR and HER2 driven pathways, of which PTEN is an important regulator. Here, we analyzed PTEN expression in 111 tissue samples of breast cancer brain metastases (BCBM). Loss of PTEN was found in a substantial proportion of BCBM samples (48.6%) and was significantly associated with triple-negative breast cancer (67.5%, p = 0.001) and a shorter survival time after surgical resection of brain metastases (p = 0.048). Overexpression of PTEN in brain-seeking MDA-MB-231 BR cells in vitro reduced activation of the AKT pathway, notably by suppression of Akt1 kinase activity. Furthermore, the migration of MDA-MB-231 BR cells in vitro was promoted by co-culturing with both astrocytes and microglial cells. Interestingly, when PTEN was overexpressed the migration was significantly inhibited. Moreover, in an ex vivo organotypic brain slice model, PTEN overexpression reduced invasion of tumor cells. This was accompanied by reduced astrocyte activation that was mediated by autocrine and paracrine activation of GM-CSF/ CSF2RA and AKT/ PTEN pathways. In conclusion, loss of PTEN is frequently detected in triple-negative BCBM patients and associated with poor prognosis. The findings of our functional studies suggest that PTEN loss promotes a feedback loop between tumor cells and glial cells, which might contribute to disease progression.

Dual Targeting of Akt and mTORC1 Impairs Repair of DNA Double-Strand Breaks and Increases Radiation Sensitivity of Human Tumor Cells.

Inhibition of mammalian target of rapamycin-complex 1 (mTORC1) induces activation of Akt. Because Akt activity mediates the repair of ionizing radiation-induced DNA double-strand breaks (DNA-DSBs) and consequently the radioresistance of solid tumors, we investigated whether dual targeting of mTORC1 and Akt impairs DNA-DSB repair and induces radiosensitization. Combining mTORC1 inhibitor rapamycin with ionizing radiation in human non-small cell lung cancer (NSCLC) cells (H661, H460, SK-MES-1, HTB-182, A549) and in the breast cancer cell line MDA-MB-231 resulted in radiosensitization of H661 and H460 cells (responders), whereas only a very slight effect was observed in A549 cells, and no effect was observed in SK-MES-1, HTB-182 or MDA-MB-231 cells (non-responders). In responder cells, rapamycin treatment did not activate Akt1 phosphorylation, whereas in non-responders, rapamycin mediated PI3K-dependent Akt activity. Molecular targeting of Akt by Akt inhibitor MK2206 or knockdown of Akt1 led to a rapamycin-induced radiosensitization of non-responder cells. Compared to the single targeting of Akt, the dual targeting of mTORC1 and Akt1 markedly enhanced the frequency of residual DNA-DSBs by inhibiting the non-homologous end joining repair pathway and increased radiation sensitivity. Together, lack of radiosensitization induced by rapamycin was associated with rapamycin-mediated Akt1 activation. Thus, dual targeting of mTORC1 and Akt1 inhibits repair of DNA-DSB leading to radiosensitization of solid tumor cells.

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.

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

Hepatocellular carcinoma (HCC) is the sixth most common cancer, and the third most common cause of cancer related death worldwide. The multi-kinase inhibitor Sorafenib represents the only systemic treatment option until today, and results from clinical trials with allosteric mTOR inhibitors were sobering. Since the PI3K/AKT/mTOR and RAF/MEK/ERK signaling pathways are frequently upregulated in HCC, we have analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY 142886) and mTOR kinase inhibitor AZD8055, given as single drugs or in combination, on proliferation and apoptosis of three HCC cell lines in vitro. We show that all three inhibitor combinations synergistically inhibit proliferation of the three HCC cell lines, with the strongest synergistic effect observed after vertical inhibition of AKT and mTORC1/2. We demonstrate that AKT kinase activity is restored 24h after blockade of mTORC1/2 by increased phosphorylation of T308, providing a rationale for combined targeting of AKT and mTOR inhibition in HCC. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with hepatocellular carcinoma.

Discontinuing MEK inhibitors in tumor cells with an acquired resistance increases migration and invasion.

BACKGROUND: Development of small molecular inhibitors against BRAF and MEK has been a breakthrough in the treatment of malignant melanoma. However, the long-term effect is foiled in virtually all patients by the emergence of resistant tumor cell populations. Therefore, mechanisms resulting in the acquired resistance against BRAF and MEK inhibitors have gained much attention and several strategies have been proposed to overcome tumor resistance, including interval treatment or withdrawal of these compounds after disease progression. METHODS: Using a panel of cell lines with an acquired resistance against MEK inhibitors, we have evaluated the sensitivity of these cells against compounds targeting AKT/mTOR signaling, as well as novel ERK1/2 inhibitors. Furthermore, the effects of withdrawal of MEK inhibitor on migration in resistant cell lines were analyzed. RESULTS: We demonstrate that withdrawal of BRAF or MEK inhibitors in tumor cells with an acquired resistance results in reactivation of ERK1/2 signaling and upregulation of EMT-inducing transcription factors, leading to a highly migratory and invasive phenotype of cancer cells. Furthermore, we show that migration in these cells is independent from AKT/mTOR signaling. However, combined targeting of AKT/mTOR using MK-2206 and AZD8055 efficiently inhibits proliferation in all resistant tumor cell lines analyzed. CONCLUSIONS: We propose that combined targeting of MEK/AKT/mTOR or treatment with a novel ERK1/2 inhibitor downstream of BRAF/MEK suppresses proliferation as well as migration and invasion in resistant tumor cells. We provide a rationale against the discontinuation of BRAF or MEK inhibitors in patients with an acquired resistance, and provide a rationale for combined targeting of AKT/mTOR and MEK/ERK1/2, or direct targeting of ERK1/2 as an effective treatment strategy.

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.

Dual Inhibition of PI3K-AKT-mTOR- and RAF-MEK-ERK-signaling is synergistic in cholangiocarcinoma and reverses acquired resistance to MEK-inhibitors.

UNLABELLED: Until today, there is no systemic treatment available for advanced cholangiocarcinoma (CCA). Recent studies have shown a frequent upregulation of the PI3K-AKT-mTOR and RAF-MEK-ERK pathways in this type of cancer. However, considering their high extend of redundancy and cross-talk, targeting only one pathway is likely to result in therapy failure and emergence of resistances. To provide a rationale for treatment of CCA with inhibitors of these respective pathways, we analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY-142886) and mTOR kinase inhibitor AZD8055 on three CCA cell lines in vitro, concerning proliferation, cell signaling and apoptosis. Furthermore, AZD6244 resistant cell lines have been generated to investigate, how their response may be affected by prolonged treatment with only a single inhibitor. Our data demonstrates that co-targeting of both, the PI3K/AKT/mTOR and RAF-MEK-ERK pathway, as well as vertical targeting of AKT and mTOR results in strong synergistic effects on proliferation and cell survival with combination indices below 0.3. Mechanistically, the combinatorial treatment with MK-2206 in addition to AZD8055 is necessary because AKT kinase activity was quickly restored after mTOR kinase inhibition. Interestingly, acquired MEK inhibitor resistance to AZD6244 was reversed by combined treatment with AZD6244 and either MK-2206 or AZD8055. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with cholangiocarcinoma. IMPLICATIONS: AKT, mTOR and MEK are promising targets for a combinatorial treatment of cholangiocarcinoma cells even after acquisition of MEK inhibitor resistance.

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.