Dynamics of Atg5-Atg12-Atg16L1 Aggregation and Deaggregation.

Macroautophagy is a physiological process that is implicated in various pathological conditions, including neurodegenerative diseases and cancer. The execution of canonical autophagy is regulated by a core signaling cascade and it involves two well-characterized, ubiquitin-like conjugation systems-the Atg5/Atg12/Atg16L1 and the Atg8-phosphatidyl ethanolamine (PE), which are both catalyzed by Atg7. The conjugation of Atg5-Atg12 and the subsequent interaction with the positive regulator Atg16L1 are essential for the conjugation of Atg8 to PE and the subsequent formation of autophagosomes. The interaction between Atg5-Atg12 complex and Atg16L1 is highly dynamic, induced upon activation of the autophagic process, and required for the recruitment of the At5-Atg12 complex to sites of autophagosome formation. Monitoring the Atg5-Atg12-Atg16L1 aggregation and deaggregation may be used not only as means to study the dynamics of autophagy, but in another important point, it may provide important insights on the basic molecular mechanisms of autophagy in physiological and pathological settings. In this chapter, we describe methods of monitoring the Atg5-Atg12-Atg16L1 aggregation and deaggregation, with emphasis on prostate cancer.

Targeting of distinct signaling cascades and cancer-associated fibroblasts define the efficacy of Sorafenib against prostate cancer cells.

Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.

Cell death induced by dexamethasone in lymphoid leukemia is mediated through initiation of autophagy.

Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. Recent studies, however, showed that an alternative mode of cell death, autophagy, is involved in the response to anticancer drugs. The specific role of autophagy and its relationship to apoptosis remains, nevertheless, controversial: it can either lead to cell survival or can function in cell death. We show that dexamethasone induced autophagy upstream of apoptosis in acute lymphoblastic leukemia cells. Inhibition of autophagy by siRNA-mediated repression of Beclin 1 expression inhibited apoptosis showing an important role of autophagy in dexamethasone-induced cell death. Dexamethasone treatment caused an upregulation of promyelocytic leukemia protein, PML, its complex formation with protein kinase B or Akt and a PML-dependent Akt dephosphorylation. Initiation of autophagy and the onset of apoptosis were both dependent on these events. PML knockout thymocytes were resistant to dexamethasone-induced death and upregulation of PML correlated with the ability of dexamethasone to kill primary leukemic cells. Our data reveal key mechanisms of dexamethasone-induced cell death that may inform the development of improved treatment protocols for lymphoid malignancies.