Browsing by Subject "Apoptosis"

Normal cells require input from extrinsic growth factors to control proliferation and survival. Recent studies have demonstrated that these same extrinsic signals also regulate cellular metabolism to ensure that metabolism adequately supports the demands of cell function, proliferation, and cell survival. The PI3K/Akt pathway is downstream of many growth factors and can promote both glucose metabolism and cell survival. Aberrant activation of the PI3K/Akt pathway is common in cancer, and its activation can contribute to the growth factor independence that is a hallmark of neoplastic cells. Metabolic demand is high in stimulated and leukemic T cells, and activation of Akt can increase glucose metabolism to meet these requirements. There is great interest in targeting the unique metabolism of cancer cells for cancer therapy, thus making an understanding of the interaction of metabolism and cell death essential.

Akt is also anti-apoptotic and can inhibit cell death by regulating members of the Bcl-2 family. Interestingly, the ability of Akt to prevent cell death is inextricably linked to its metabolic function. Several recent studies have demonstrated that glucose metabolism can affect Bcl-2 to family members to promote cell survival, but the role of Akt-dependent glucose metabolism in the regulation of Bcl-2 family members is not understood. Using a model of growth factor withdrawal-induced apoptosis, we show that Akt prevents cell death by maintaining glucose metabolism to regulate the Bcl-2 family members Puma and Mcl-1, and demonstrate the importance of this pathway in the survival of stimulated T lymphocytes and leukemia.

After growth factor withdrawal, active Akt suppressed Puma induction in abundant glucose, but Puma was rapidly upregulated in glucose-deficient conditions and was necessary and sufficient to promote efficient cell death. Importantly, glucose was not uniquely required, as provision of alternative mitochondrial fuels allowed Akt to suppress Puma and maintain survival. This metabolic regulation of Puma was mediated through partially p53-dependent transcriptional induction as well as control of Puma protein stability.

In addition to inhibiting Puma expression, active Akt prevented the loss of Mcl-1 after growth factor withdrawal by sustaining Mcl-1 protein synthesis in a glucose-dependent manner. Mcl-1 was essential for preventing Bim-induced apoptosis, as Akt could not inhibit Bim induction after growth factor deprivation. Slowing of Mcl-1 synthesis by inhibiting glucose metabolism reversed Mcl-1-mediated resistance of leukemic cells to the Bcl-2 inhibitor ABT-737. Importantly, Akt and glucose-reliant Mcl-1 expression required mTOR-dependent phosphorylation of 4EBP, and treatment with mTOR inhibitors also reversed ABT-737 resistance.

Together, this study demonstrates that Akt promotes cell survival by preventing metabolic checkpoints that stimulate Puma expression and stability and inhibit Mcl-1 synthesis, advancing our understanding of the links between metabolism and cell death. These studies highlight the importance of cellular metabolism--including a potential role for the alternative sugar fructose--in cancer cell survival that may provide a mechanistic understanding to drive development of metabolism-targeted cancer therapies.

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. Although pyroptosis and apoptosis have distinct signaling pathways, when a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we also now engineer S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. In both engineered strains, apoptosis occurred somewhat slower than pyroptosis. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche, but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. In order to clear an infection, distinct cell types may have specific tasks that they must complete before they die. In some cells, either apoptotic or pyroptotic signaling may initiate the same tasks, whereas in other cell types these modes of cell death may lead to different tasks that may not be identical in defense against infection. We recently suggested that such diverse tasks can be considered as different cellular “bucket lists” to be accomplished before a cell dies. As demonstrated here, engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We engineer S. Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. However, after 48 hours the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFNg-deficient mice this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell’s physiology that make host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Cancer cells, however, can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells are deprived sufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and cells activate the self-digestive process of autophagy, which clears damaged organelles and provides degradation products as an alternate fuel to support mitochondrial metabolism. Importantly, loss of growth signals can also lead to apoptosis mediated through Bcl-2 family proteins, and Bcl-2 has been reported to interfere with autophagy, potentially disrupting a key nutrient source just as glucose uptake becomes limiting. Since autophagy may support survival or lead to death depending on context, the role of this pathway in apoptosis-competent growth factor deprived cells remains unclear.

In this thesis, I examine the interactions of autophagy with Bcl-2 family proteins and apoptosis upon inhibition of growth signals in hematopoietic cells. In contrast to other studies, I found autophagy was rapidly induced in growth factor deprived cells regardless of Bcl-2 or Bcl-xL expression, and this led to increased production of fatty acids and amino acids for metabolism. While these data suggested autophagy may play a key role to support metabolism of growth factor deprived cells, provision of exogenous pyruvate or lipids as alternate fuel had little affect on cell survival. Instead, I found that autophagy modulated cell stress pathways and Bcl-2 family protein expression in a context specific fashion to impact cell fate.

My results show that autophagy's effect on cell survival is dependent on its level of induction within a cell. I observed that partial suppression of autophagy protects cells from stress and induction of pro-apoptotic Bcl-2 family expression, while complete inhibition of autophagy enhances stress and is pro-apoptotic. In experiments using shRNAi to partially suppress autophagy, I found increased survival upon growth factor deprivation in several different types of cells expressing anti-apoptotic Bcl-2 or Bcl-xL, indicating that autophagy promoted cell death in these instances. Cell death was not autophagic, but apoptotic, and relied on direct Chop-dependent transcriptional induction of the pro-apoptotic Bcl-2 family protein Bim. In contrast, complete acute disruption of autophagy through conditional Cre-mediated excision of the autophagy-essential gene Atg3 led to p53 phosphorylation, upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma, and rapid cell death of cells the presence or absence of growth factor. Importantly, transformed BCR-Abl-expressing cells had low basal levels of autophagy but were highly dependent on this process. Deletion of Atg3 or treatment with chemical autophagy inhibitors led to rapid apoptosis, and BCR-Abl expressing cells were unable to form leukemia in mice in without autophagy. Together, my data demonstrate a dual role for autophagy in cell survival or cell death and suggest that the level of autophagy in a cell is critical in determining its role in apoptosis and cell fate. Ultimately, these results may help to determine future approaches to modulate autophagy in cancer therapy.

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