Alexander A. Goldberg

A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane

We define the dynamics of spatial and temporal reorganization of the team of proteins and lipids serving peroxisome division. The peroxisome becomes competent for division only after it acquires the complete set of matrix proteins involved in lipid metabolism. Overloading the peroxisome with matrix proteins promotes the relocation of acyl-CoA oxidase (Aox), an enzyme of fatty acid β-oxidation, from the matrix to the membrane. The binding of Aox to Pex16p, a membrane-associated peroxin required for peroxisome biogenesis, initiates the biosynthesis of phosphatidic acid and diacylglycerol (DAG) in the membrane. The formation of these two lipids and the subsequent transbilayer movement of DAG initiate the assembly of a complex between the peroxins Pex10p and Pex19p, the dynamin-like GTPase Vps1p, and several actin cytoskeletal proteins on the peroxisomal surface. This protein team promotes membrane fission, thereby executing the terminal step of peroxisome division.

Bile acids induce apoptosis selectively in androgen-dependent and -independent prostate cancer cells

Prostate cancer is a prevalent age-related disease in North America, accounting for about 15% of all diagnosed cancers. We have previously identified lithocholic acid (LCA) as a potential chemotherapeutic compound that selectively kills neuroblastoma cells while sparing normal human neurons. Now, we report that LCA inhibits the proliferation of androgen-dependent (AD) LNCaP prostate cancer cells and that LCA is the most potent bile acid with respect to inducing apoptosis in LNCaP as well as androgen-independent (AI) PC-3 cells, without killing RWPE-1 immortalized normal prostate epithelial cells. In LNCaP and PC-3 cells, LCA triggered the extrinsic pathway of apoptosis and cell death induced by LCA was partially dependent on the activation of caspase-8 and -3. Moreover, LCA increased cleavage of Bid and Bax, down-regulation of Bcl-2, permeabilization of the mitochondrial outer membrane and activation of caspase-9. The cytotoxic actions of LCA occurred despite the inability of this bile acid to enter the prostate cancer cells with about 98% of the nominal test concentrations present in the extracellular culture medium. With our findings, we provide evidence to support a mechanism of action underlying the broad anticancer activity of LCA in various human tissues.

Mechanisms through which lithocholic acid delays yeast chronological aging under caloric restriction conditions

All presently known geroprotective chemical compounds of plant and microbial origin are caloric restriction mimetics because they can mimic the beneficial lifespan- and healthspan-extending effects of caloric restriction diets without the need to limit calorie supply. We have discovered a geroprotective chemical compound of mammalian origin, a bile acid called lithocholic acid, which can delay chronological aging of the budding yeast Saccharomyces cerevisiae under caloric restriction conditions. Here, we investigated mechanisms through which lithocholic acid can delay chronological aging of yeast limited in calorie supply. We provide evidence that lithocholic acid causes a stepwise development and maintenance of an aging-delaying cellular pattern throughout the entire chronological lifespan of yeast cultured under caloric restriction conditions. We show that lithocholic acid stimulates the aging-delaying cellular pattern and preserves such pattern because it specifically modulates the spatiotemporal dynamics of a complex cellular network. We demonstrate that this cellular network integrates certain pathways of lipid and carbohydrate metabolism, some intercompartmental communications, mitochondrial morphology and functionality, and liponecrotic and apoptotic modes of aging-associated cell death. Our findings indicate that lithocholic acid prolongs longevity of chronologically aging yeast because it decreases the risk of aging-associated cell death, thus increasing the chance of elderly cells to survive.

Abstract 3298: Diindolylmethane and its halogenated analogues induce autophagy in human prostate cancer cells via induction of the astrocyte-elevated gene-1 (AEG-1)

3,3’-diindolylmethane (DIM) and its halogenated derivatives (ring-DIMs) are recently shown to induce protective autophagy in human prostate cancer cells. The mechanism of induction of autophagy by DIM has not been elucidated. As DIM is a mitochondrial ATP synthase inhibitor, we hypothesized that DIM and ring-DIMs induce autophagy via alteration of AMP/ATP ratio and activation of AMPK signaling in prostate cancer cells. Autophagic activity was monitored by LC3B-I to LC3B-II conversion in LNCaP and C42B prostate cancer cells. Autophagic vacuoles were examined using Cyto-ID autophagy detection kit and transmission electron microscope (TEM). Protein levels for AMPK, pAMPK, acetyl-CoA carboxylase (ACC), pACC, AEG-1, pULK-1 and β-actin were measured by western blot. AMPK and AEG-1 gene expression was downregulated using siRNA. DIM and ring-DIMs induced autophagy by increasing autophagic vacuoles and LC3B-I to LC3B-II conversion in LNCaP and C42B cells. These compounds also induced AMPK, ULK-1 and ACC phosphorylation in a time dependent manner. Interestingly, DIM, 4,4’ dibromoDIM and 7,7’ dichloroDIM induced the oncogenic protein AEG-1 time dependently in LNCaP and C42B cells. Downregulation of AEG-1 or AMPK inhibited DIM- and ring-DIM-induced autophagy. Pretreatment with ULK1 inhibitor MRT 67307 or siRNAs targeting AEG-1 or AMPK potentiated the cytotoxicity of DIM and ring-DIMs. Interestingly, downregulation of AEG-1 induced senescence in cells treated with toxic concentrations of DIM or ring-DIMs, and inhibited the onset of apoptosis in response to DIM or ring-DIMs. In summary, we identified a novel mechanism for DIM- and ring-DIM-induced protective autophagy, via induction of AEG-1 and activation of AMPK. Our findings could help towards the development of novel drug therapies for prostate cancer that include selective autophagy inhibitors as adjuvants. Citation Format: Hossam Draz, Alexander Goldberg, Thomas Sanderson, Stephen Safe. Diindolylmethane and its halogenated analogues induce autophagy in human prostate cancer cells via induction of the astrocyte-elevated gene-1 (AEG-1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3298. doi:10.1158/1538-7445.AM2017-3298

Oncogenic effects of urotensin-II in cells lacking tuberous sclerosis complex-2

Lymphangioleiomyomatosis (LAM) is a destructive lung disease that can arise sporadically or in adults suffering from the tumor syndrome tuberous sclerosis complex (TSC). Microscopic tumors (‘LAM nodules’) in the lung interstitium arise from lymphatic invasion and metastasis. These consist of smooth muscle-like cells (LAM cells) that exhibit markers of neural crest differentiation and loss of the tumor suppressor protein ‘tuberous sclerosis complex-2’ (TSC2). Consistent with a neural phenotype, expression of the neuropeptide urotensin-II and its receptor was detected in LAM nodules. We hypothesized that loss of TSC2 sensitizes cells to the oncogenic effects of urotensin-II. TSC2-deficient Eker rat uterine leiomyoma ELT3 cells were stably transfected with empty vector or plasmid for the expression of TSC2. Urotensin-II increased cell viability and proliferation in TSC2-deficient cells, but not in TSC2-reconstituted cells. When exposed to urotensin-II, TSC2-deficient cells exhibited greater migration, anchorage-independent cell growth, and matrix invasion. The effects of urotensin-II on TSC2-deficient cells were blocked by the urotensin receptor antagonist SB657510, and accompanied by activation of Erk mitogen-activated protein kinase and focal adhesion kinase. Urotensin-II-induced proliferation and migration were reproduced in TSC2-deficient human angiomyolipoma cells, but not in those stably expressing TSC2. In a mouse xenograft model, SB657510 blocked the growth of established ELT3 tumors, reduced the number of circulating tumor cells, and attenuated the production of VEGF-D, a clinical biomarker of LAM. Urotensin receptor antagonists may be selective therapeutic agents for the treatment of LAM or other neural crest-derived neoplasms featuring loss of TSC2 or increased expression of the urotensin receptor.

Abstract B256: Ring-DIMs induce mitochondrial dysfunction and ER stress in human prostate cancer cells.

We have previously shown that a series of brominated and chlorinated analogs of 3,39-diindolylmethane (DIM) can inhibit can induce apoptosis and necrosis in androgen-dependent and androgen-independent prostate cancer cells and that addition of bromine to the 4 and 4’ position of the indole ring of DIM maximally increases the potency of the anticancer compound. To understand the upstream events leading to the activation of caspases in response to treatment with ring-DIMs in androgen-dependent LNCaP and androgen-independent LNCaP C4-2B cells by monitoring the onset of endoplasmic reticulum (ER) stress and the dysregulation of mitochondrial respiration. We found that 4,49-dibromo- and 7,79-dichloroDIM and DIM itself induced ER stress-dependent upregulation of CHOP, ATF4, and GRP78, while only 4,49-dibromo and 7,79-dichloroDIM induced phosphorylation of eIF2alpha and JNK. Both ER stress and loss of mitochondrial membrane potential were observed after treatment with 4,49-dihaloDIMs and DIM, but not 7,79-dihaloDIMs within 1 hour of exposure, before the appearance of later stage apoptotic events such as condensed chromatin. Salubrinal inhibited cell death induced by 4,49-dihaloDIMs, but facilitated cell death induced by 7,79-dihaloDIMs or DIM. Interestingly, salubrinal did not increase eIF2alpha phosphorylation after co-treatment with either DIM or ring-DIMs. However, it did restore mitochondrial membrane potential in cells treated with 4,49dihaloDIMs and further decreased mitochondrial activity after co-treatment with 7,79-dihaloDIMs or DIM. Moreover, cyclosporin A inhibited cell death induced by both 4,4’-dihalo and 7,7’-dihaloDIMs but not DIM. Taken together, these data suggest that the ring-DIMs induce cell death via mitochondrial dysfunction and ER stress, and that because salubrinal either stimulates or inhibits cell death in combination with specific ring-DIMs, its effects are related to mitochondrial membrane integrity and not to phosphorylation of eIF2alpha. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B256. Citation Format: Alexander A. Goldberg, Diana Montes-Grajales, Jesus Olivero, Adam Beach, Vladimir Titorenko, Steven Safe, Thomas Sanderson. Ring-DIMs induce mitochondrial dysfunction and ER stress in human prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B256.