Search

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, remains the second leading cause of cancer-related death. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP-531. We report potent anti-tumor effects of unacylated ghrelin, dependent on cells being cultured in 3D in a biologically-relevant extracellular matrix. The mechanism of unacylated ghrelin-mediated growth inhibition involves activation of Gαi and suppression of MAPK signaling. AZP-531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, it remains the second leading cause of cancer-related death in the US. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. One major area of research relates to its reported effects to increase insulin sensitivity in diabetics, this being the basis for the development of unacylated ghrelin analog, AZP531 or livoletide, now in clinical trials for the treatment of Prader-Willi Syndrome. The mechanism of action of unacylated ghrelin is largely uncharacterized, in any system, because it does not bind to or activate the cognate ghrelin receptor, GHSR1a. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP531. We report potent effects of unacylated ghrelin, at picomolar doses, on the growth of breast cancer cells, dependent on 3D culture and activation of Gαi. Suppression of MAPK signaling by unacylated ghrelin leads to cell cycle arrest and apoptosis. AZP531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that are related genetically and pathologically. Mutations in the UBQLN2 gene, encoding the ubiquitin-like protein ubiquilin2, are associated with familial ALS/FTD, but the pathophysiological mechanisms remain unclear. Here, we demonstrate that ALS/FTD UBQLN2 mutants P497H and P506T inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in neuronal cells. In addition, we observed that Sec31-positive ER exit sites are clustered in UBQLN2T487I patient spinal cord tissues. Both the ER-Golgi intermediate (ERGIC) compartment and the Golgi become disorganised and fragmented. This activates ER stress and inhibits ER-associated degradation. Hence, this study highlights perturbations in secretory protein trafficking and ER homeostasis as pathogenic mechanisms associated with ALS/FTD-associated forms of UBQLN2.

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.

Amyotrophic lateral sclerosis (ALS) is characterised by the formation of intracellular misfolded protein inclusions that form in motor neurons. Autophagy is the major degradation pathway for aggregate-prone proteins within lysosomes. Autophagy begins by the production of the omegasome, forming the autophagosome membrane, which then fuses with the lysosome. Mutations in fused in sarcoma (FUS) cause 5% of familial ALS cases and FUS-positive inclusions are also formed in sporadic ALS tissues. In this study, we demonstrate that the expression of ALS-associated mutant FUS impairs autophagy in neuronal cells. In mutant FUS-expressing neuronal cells, accumulation of ubiquitinated proteins and autophagy substrates p62 and NBR1 was detected, and formation of both the omegasome and autophagosome was inhibited in these cells. However, overexpression of Rab1 rescued these defects, suggesting that Rab1 is protective in ALS. The number of LC3-positive vesicles was also increased in motor neurons from the spinal cord of an ALS patient carrying a FUS (R521C) mutation compared with a control patient, providing additional evidence that autophagy is dysregulated in mutant FUS-associated ALS. This study provides further understanding of the intricate autophagy system and neurodegeneration in ALS.

Cu/Zn-superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER-Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER-Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER-Golgi transport by over-expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER-Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.

Mutations in Cu, Zn-superoxide dismutase 1 (SOD1) are associated with degeneration of motor neurons in the disease, familial amyotrophic lateral sclerosis. Intracellular protein inclusions containing mutant SOD1 (mSOD1) are associated with disease but it is unclear whether they are neuroprotective or cytotoxic. We report here that the formation of mSOD1 inclusions in a motor neuron-like cell line (NSC-34) strongly correlates with apoptosis via the mitochondrial death pathway. Applying confocal microscopic analyses, we observed changes in nuclear morphology and activation of caspase 3 specifically in cells expressing mSOD1 A4V or G85R inclusions. Furthermore, markers of mitochondrial apoptosis (activation and recruitment of Bax, and cytochrome c redistribution) were observed in 30% of cells bearing mSOD1 inclusions but not in cells expressing dispersed SOD1. In the presence of additional apoptotic challenges (staurosporine, etoposide, and hydrogen peroxide), cells bearing mSOD1 inclusions were susceptible to further apoptosis suggesting they were in a pro-apoptotic state, thus confirming that inclusions are linked to toxicity. Surprisingly, cells displaying dispersed SOD1 [both wildtype (WT) and mutant] were protected against apoptosis upstream of mitochondrial apoptotic signaling, induced by all agents tested. This protection against apoptosis was unrelated to SOD1 enzymatic activity because the G85R that lacks enzymatic function protected cells similarly to both WT SOD1 and A4V that possesses WT-like activity. These findings demonstrate new aspects of SOD1 in relation to cellular viability; specifically, mSOD1 can be either neuroprotective or cytotoxic depending on its aggregation state.