Tuberous sclerosis complex (TSC) disease results from inactivation of the TSC1 or TSC2 gene, and is characterized by benign tumors in several organs. Because TSC tumorigenesis correlates with hyperactivation of mTORC1, current therapies focus on mTORC1 inhibition with rapamycin or its analogs. Rapamycin-induced tumor shrinkage has been reported, but tumor recurrence occurs on withdrawal from rapamycin. Autophagy has been associated with development of TSC tumors and with tumor cell survival during rapamycin treatment. mTORC1 and AMPK directly inhibit and activate autophagy, respectively. AMPK is hyperactivated in TSC cells and tumors, and drives cytoplasmic sequestration of the cell-cycle inhibitor p27KIP (p27). Whether AMPK and p27 are involved in rapamycin-induced autophagy and survival of TSC cells remain unexplored. Here, we show that inhibition of AMPK by compound C or by shRNA-mediated depletion of LKB1 reduces activation of autophagy by rapamycin in Tsc2-null cells. Similarly, shRNA-mediated depletion of p27 inhibited rapamycin-induced autophagy. In support of p27 lying downstream of AMPK on the activation of autophagy in Tsc2-null cells, a p27 mutant that preferentially localizes in the cytosol recovered the effect of rapamycin on autophagy in both p27- and LKB1-depleted cells, but a nuclear p27 mutant was inactive. Finally, we show that p27-dependent activation of autophagy is involved in Tsc2-null cell survival under rapamycin treatment. These results indicate that an AMPK/p27 axis is promoting a survival mechanism that could explain in part the relapse of TSC tumors treated with rapamycin, exposing new avenues for designing more efficient treatments for TSC patients.

Currently, gastric cancer (GC) and gallbladder cancer (GBC) constitute important causes of human deaths related to cancer worldwide. In the last years, several researches are focused on the role of dietary compounds in preventing cancers. The consumption of fruits with high antioxidants, mainly anthocyanins, represents a good option to reduce the risk of chronic human diseases. Calafate (Berberis microphylla G. Forst.) berries, recognized by their remarkable antioxidant properties and high content of anthocyanins, appear as a new alternative to treat degenerative diseases of public interest. The present work was aimed to evaluate the impact of crude and anthocyanin-rich extracts from Calafate fruits on in vitro cell viability and migration capacity of gastric (AGC) and gallbladder (G415) human cancer cell lines, as related with their antioxidant properties. Crude and anthocyanin-rich extracts were obtained from fruits of Calafate grown under field conditions in the south of Chile. Antioxidants, phenols, anthocyanins, and anthocyanidins were determined. In vitro cell viability and migration of AGS and G415 human cancer cell lines at different concentrations of extracts (25-800 μg mL −1) were determined. Anthocyanin-rich extracts of Calafate berries showed comparable antioxidant activity (up to 1200 μg Trolox eq. g −1 DW), slightly lower total phenolic content (12%), but higher total anthocyanin content (25%) compared to the crude extract. The major anthocyanidin molecule detected in both extracts was delphinidin, followed by malvidin, and low concentrations of petunidin, cyanidin, and peonidin. As expected, all of these compounds were detected in higher levels in anthocyanin-rich extracts (up to 2-fold). Noteworthy, our study revealed that Calafate fruit extracts strongly decrease in vitro viability and migration capacity of gastric carcinoma (AGC model) and gallbladder carcinoma (G415 model) human cell lines; however, the anthocyanin-rich extract displayed higher inhibitory effects (up to 70%) compared to crude extracts. These findings allow suggesting that the in vitro antiproliferative potential of Calafate fruit extract is strongly related to the anthocyanin concentration, especially delphinidin.

Rheb is a small GTPase member of the Ras superfamily and an activator of mTORC1, a protein complex master regulator of cell metabolism, growth, and proliferation. Rheb/mTORC1 pathway is hyperactivated in proliferative diseases, such as Tuberous Sclerosis Complex syndrome and cancer. Therefore, targeting Rheb-dependent signaling is a rational strategy for developing new drug therapies. Rheb activates mTORC1 in the cytosolic surface of lysosomal membranes. Rheb’s farnesylation allows its anchorage on membranes, while its proper localization depends on the prenyl-binding chaperone PDEδ. Recently, the use of PDEδ inhibitors has been proposed as anticancer agents because they interrupted KRas signaling leading to antiproliferative effects in KRas-dependent pancreatic cancer cells. However, the effect of PDEδ inhibition on the Rheb/mTORC1 pathway has been poorly investigated. Here, we evaluated the impact of a new PDEδ inhibitor, called Deltasonamide 1, in Tsc2-null MEFs, a Rheb-dependent overactivated mTORC1 cell line. By using a yeast two-hybrid assay, we first validated that Deltasonamide 1 disrupts Rheb-PDEδ interaction. Accordingly, we found that Deltasonamide 1 reduces mTORC1 targets activation. In addition, our results showed that Deltasonamide 1 has antiproliferative and cytotoxic effects on Tsc2-null MEFs but has less effect on Tsc2-wild type MEFs viability. This work proposes the pharmacological PDEδ inhibition as a new approach to target the abnormal Rheb/mTORC1 activation in Tuberous Sclerosis Complex cells.

Cancer cells evade cell death during tumor progression under metabolic stress. However, molecular mechanisms involved in cellular adaptation to this stress are still poorly understood. NUAK1, a serine threonine kinase of the AMPK catalytic subunit family, has been involved in tumor cell survival, but its regulation and function under metabolic stress remain unclear. For this study, we have used mouse embryo fibroblasts (MEFs) and cancer cells to better understand the role of NUAK1 in cell survival. We found that NUAK1 mRNA and protein levels increase in response to metabolic stress, suggesting a transcriptional regulation of NUAK1 expression. In addition, we found that NUAK1 is expressed almost exclusively in the nucleus of MEFs and several cancer cell lines. Because of the nuclear localization of NUAK1 and previous studies showing that NUAK1 regulates p53 transcription factor, we investigated whether p53 is necessary for NUAK1´s effect on cell survival. By using p53ER/ER MEFs that can be reversibly and rapidly switched between p53 wild-type and knockout states, we demonstrated that NUAK1 can promote cell survival in a p53-independent cellular context. Furthermore, NUAK1 was involved in cell survival of p53-null H1299 lung cancer and p53-null HCT116 colorectal cancer cells under metabolic stress. In parallel to the increase of NUAK1 expression, we found that NUAK1 is phosphorylated. LKB1 kinase is known to phosphorylate NUAK1, increasing NUAK1 kinase activity. However, shRNA-mediated depletion of LKB1 or mutation of the NUAK1 residue phosphorylated by LKB1 did not affect NUAK1 function on cell survival. By bioinformatics analysis of NUAK1 protein sequence, we found several sites predicted to be phosphorylated by JNK and p38. Future studies will determine whether JNK or p38 is involved in NUAK1 phosphorylation, and also evaluate whether NUAK1 nuclear localization is directly related with tumor progression in a p53-independent manner. Overall, our results showed that NUAK1 is transcriptional and post-translational regulated in response to metabolic stress, and that the effect of NUAK1 on cell survival is independently of LKB1 and p53. Funded by FONDECYT 1120923 Citation Format: Mario Palma, Daniela Peña, Katherine Venturelli, Ariel F. Castro. Nuclear NUAK1 function on cancer cell survival, independently of LKB1 and p53. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A36.