Suppression of autophagy impedes glioblastoma development and induces senescence.

The function of macroautophagy/autophagy during tumor initiation or in established tumors can be highly distinct and context-dependent. To investigate the role of autophagy in gliomagenesis, we utilized a KRAS-driven glioblastoma mouse model in which autophagy is specifically disrupted via RNAi against Atg7, Atg13 or Ulk1. Inhibition of autophagy strongly reduced glioblastoma development, demonstrating its critical role in promoting tumor formation. Further supporting this finding is the observation that tumors originating from Atg7-shRNA injections escaped the knockdown effect and thereby still underwent functional autophagy. In vitro, autophagy inhibition suppressed the capacity of KRAS-expressing glial cells to form oncogenic colonies or to survive low serum conditions. Molecular analyses revealed that autophagy-inhibited glial cells were unable to maintain active growth signaling under growth-restrictive conditions and were prone to undergo senescence. Overall, these results demonstrate that autophagy is crucial for glioma initiation and growth, and is a promising therapeutic target for glioblastoma treatment.

Endogenous human MDM2-C is highly expressed in human cancers and functions as a p53-independent growth activator.

Human cancers over-expressing mdm2, through a T to G variation at a single nucleotide polymorphism at position 309 (mdm2 SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, mdm2-C. Alternatively spliced mdm2 transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells. However no study to date has detected endogenous MDM2 protein isoforms. Studies with exogenous expression of splice variants have been carried out with mdm2-A and mdm2-B, but the mdm2-C isoform has remained virtually unexplored. We addressed the cellular influence of exogenously expressed MDM2-C, and asked if endogenous MDM2-C protein was present in human cancers. To detect endogenous MDM2-C protein, we created a human MDM2-C antibody to the splice junction epitope of exons four and ten (MDM2 C410) and validated the antibody with in vitro translated full length MDM2 compared to MDM2-C. Interestingly, we discovered that MDM2-C co-migrates with MDM2-FL at approximately 98 kDa. Using the validated C410 antibody, we detected high expression of endogenous MDM2-C in human cancer cell lines and human cancer tissues. In the estrogen receptor positive (ER+) mdm2 G/G SNP309 breast cancer cell line, T47D, we observed an increase in endogenous MDM2-C protein with estrogen treatment. MDM2-C localized to the nucleus and the cytoplasm. We examined the biological activity of MDM2-C by exogenously expressing the protein and observed that MDM2-C did not efficiently target p53 for degradation or reduce p53 transcriptional activity. Exogenous expression of MDM2-C in p53-null human cancer cells increased colony formation, indicating p53-independent tumorigenic properties. Our data indicate a role for MDM2-C that does not require the inhibition of p53 for increasing cancer cell proliferation and survival.

The ULK1 complex: sensing nutrient signals for autophagy activation.

The Atg1/ULK1 complex plays a central role in starvation-induced autophagy, integrating signals from upstream sensors such as MTOR and AMPK and transducing them to the downstream autophagy pathway. Much progress has been made in the last few years in understanding the mechanisms by which the complex is regulated through protein-protein interactions and post-translational modifications, providing insights into how the cell modulates autophagy, particularly in response to nutrient status. However, how the ULK1 complex transduces upstream signals to the downstream central autophagy pathway is still unclear. Although the protein kinase activity of ULK1 is required for its autophagic function, its protein substrate(s) responsible for autophagy activation has not been identified. Furthermore, examples of potential ULK1-independent autophagy have emerged, indicating that under certain specific contexts, the ULK1 complex might be dispensable for autophagy activation. This raises the question of how the autophagic machinery is activated independent of the ULK1 complex and what are the biological functions of such noncanonical autophagy pathways.

Role of autophagy in histone deacetylase inhibitor-induced apoptotic and nonapoptotic cell death.

Autophagy is a cellular catabolic pathway by which long-lived proteins and damaged organelles are targeted for degradation. Activation of autophagy enhances cellular tolerance to various stresses. Recent studies indicate that a class of anticancer agents, histone deacetylase (HDAC) inhibitors, can induce autophagy. One of the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), is currently being used for treating cutaneous T-cell lymphoma and under clinical trials for multiple other cancer types, including glioblastoma. Here, we show that SAHA increases the expression of the autophagic factor LC3, and inhibits the nutrient-sensing kinase mammalian target of rapamycin (mTOR). The inactivation of mTOR results in the dephosphorylation, and thus activation, of the autophagic protein kinase ULK1, which is essential for autophagy activation during SAHA treatment. Furthermore, we show that the inhibition of autophagy by RNAi in glioblastoma cells results in an increase in SAHA-induced apoptosis. Importantly, when apoptosis is pharmacologically blocked, SAHA-induced nonapoptotic cell death can also be potentiated by autophagy inhibition. Overall, our findings indicate that SAHA activates autophagy via inhibiting mTOR and up-regulating LC3 expression; autophagy functions as a prosurvival mechanism to mitigate SAHA-induced apoptotic and nonapoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of SAHA.