Novel flexible heteroarotinoid, SL-1-39, inhibits HER2-positive breast cancer cell proliferation by promoting lysosomal degradation of HER2.

SL-1-39 [1-(4-chloro-3-methylphenyl)-3-(4-nitrophenyl)thiourea] is a new flexible heteroarotinoid (Flex-Het) analog derived from the parental compound, SHetA2, previously shown to inhibit cell growth across multiple cancer types. The current study aims to determine growth inhibitory effects of SL-1-39 across the different subtypes of breast cancer cells and delineate its molecular mechanism. Our results demonstrate that while SL-1-39 blocks cell proliferation of all breast cancer subtypes tested, it has the highest efficacy against HER2+ breast cancer cells. Molecular analyses suggest that SL-1-39 prevents S phase progression of HER2+ breast cancer cells (SKBR3 and MDA-MB-453), which is consistent with reduced expression of key cell-cycle regulators at both the protein and transcriptional levels. SL-1-39 treatment also decreases the protein levels of HER2 and pHER2 as well as its downstream effectors, pMAPK and pAKT. Reduction of HER2 and pHER2 at the protein level is attributed to increased lysosomal degradation of total HER2 levels. This is the first study to show that a flexible heteroarotinoid analog modulates the HER2 signaling pathway through lysosomal degradation, and thus further warrants the development of SL-1-39 as a therapeutic option for HER2+ breast cancer.

Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease.

Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.

Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members.

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.

Endogenously EGFP-Labeled Mouse Embryonic Stem Cells.

Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping ß-actin promoter The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759-769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies.

A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.

Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor γ coactivator (PGC)-1α, PGC-1ß and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.

Valosin-containing protein gene mutations: cellular phenotypes relevant to neurodegeneration.

Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.

A calpain-like protease inhibits autophagic cell death.

Programmed cell death (PCD) plays critical roles during development and in disease states. One form of programmed cell death utilizes autophagy--a cellular mechanism of degrading bulk cytosolic components--to destroy cells. Previously, the broad-spectrum caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) was shown to induce autophagic cell death. The mechanism of Zvad-induced cell death was proposed to require caspase-8 inhibition. In our report, we extend these findings to show that--as is the case for apoptosis--induction of autophagic cell death in response to zVAD results in phosphatidylserine exposure prior to loss of membrane integrity. Additionally, we show that caspase-8 inhibition is insufficient to cause autophagic cell death. Rather, the activity of a calpain-like protease must also be blocked. These results reveal the existence of an autophagic PCD-inhibiting calpain-like cysteine protease.