8p22 MTUS1 Gene Product ATIP3 Is a Novel Anti-Mitotic Protein Underexpressed in Invasive Breast Carcinoma of Poor Prognosis

Background

Breast cancer is a heterogeneous disease that is not totally eradicated by current therapies. The classification of breast tumors into distinct molecular subtypes by gene profiling and immunodetection of surrogate markers has proven useful for tumor prognosis and prediction of effective targeted treatments. The challenge now is to identify molecular biomarkers that may be of functional relevance for personalized therapy of breast tumors with poor outcome that do not respond to available treatments. The Mitochondrial Tumor Suppressor (MTUS1) gene is an interesting candidate whose expression is reduced in colon, pancreas, ovary and oral cancers. The present study investigates the expression and functional effects of MTUS1 gene products in breast cancer.

Methods and Findings

By means of gene array analysis, real-time RT-PCR and immunohistochemistry, we show here that MTUS1/ATIP3 is significantly down-regulated in a series of 151 infiltrating breast cancer carcinomas as compared to normal breast tissue. Low levels of ATIP3 correlate with high grade of the tumor and the occurrence of distant metastasis. ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy. Functional studies indicate that silencing ATIP3 expression by siRNA increases breast cancer cell proliferation. Conversely, restoring endogenous levels of ATIP3 expression leads to reduced cancer cell proliferation, clonogenicity, anchorage-independent growth, and reduces the incidence and size of xenografts grown in vivo. We provide evidence that ATIP3 associates with the microtubule cytoskeleton and localizes at the centrosomes, mitotic spindle and intercellular bridge during cell division. Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis.

Conclusions

Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome. ATIP3 re-expression limits tumor cell proliferation in vitro and in vivo, suggesting that this protein may represent a novel useful biomarker and an interesting candidate for future targeted therapies of aggressive breast cancer.     

A conserved sequence immediately N-terminal to the Bateman domains in AMP-activated protein kinase gamma subunits is required for the interaction with the beta subunits

Mammalian AMP-activated protein kinase is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMP-activated protein kinase is a heterotrimer of three different subunits, i.e. alpha, beta, and gamma, with alpha being the catalytic subunit and beta and gamma having regulatory roles. Although several studies have defined different domains in alpha and beta involved in the interaction with the other subunits of the complex, little is known about the regions of the gamma subunits involved in these interactions. To study this, we have made sequential deletions from the N termini of the gamma subunit isoforms and studied the interactions with alpha and beta subunits, both by two-hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20-25 amino acids in gamma1, gamma2, and gamma3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active alphabetagamma complex. This region is required for the interaction with the beta subunits. The interaction between the alpha and gamma subunits does not require this region and occurs instead within the Bateman domains of the gamma subunit, although the alpha-gamma interaction does appear to stabilize the beta-gamma interaction. In addition, sequential deletions from the C termini of the gamma subunits indicate that deletion of any of the CBS (cystathionine beta-synthase) motifs prevents the formation of a functional complex with the alpha and beta subunits.     

Intraventricular (i3vt) ghrelin increases food intake in fatty Zucker rats

Ghrelin is an orexigenic peptide secreted from the stomach and also made in the brain. Ghrelin receptors are expressed on hypothalamic cells important in appetite and energy balance. We determined that intra-3rd-ventricular (i3vt) ghrelin dose-dependently increases acute (1 and 2 h) food intake in lean and fatty Zucker rats (0, 0.01, 0.1 and 1.0 nmol ghrelin). The percentage increase of food intake in fatty Zucker rats was significantly greater than that in lean rats. Fatty Zucker rats had 4.5 times more ghrelin receptor mRNA in the hypothalamus than lean Zucker rats, suggesting a possible mechanism for the increased sensitivity.     

Leaching of contaminated leaves following uptake and phytoremediation of RDX, HMX, and TNT by poplar

The uptake and fate of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by hybrid poplars in hydroponic systems were compared and exposed leaves were leached with water to simulate potential exposure pathways from groundwater in the field. TNT was removed from solution more quickly than nitramine explosives. Most of radioactivity remained in root tissues for 14C-TNT, but in leaves for 14C-RDX and 14C-HMX. Radiolabel recovery for TNT and HMX was over 94%, but that of RDX decreased over time, suggesting a loss of volatile products. A considerable fraction (45.5%) of radioactivity taken up by whole plants exposed to 14C-HMX was released into deionized water, mostly as parent compound after 5 d of leaching. About a quarter (24.0%) and 1.2% were leached for RDX and TNT, respectively, mostly as transformed products. Leached radioactivity from roots was insignificant in all cases (< 2%). This is the first report in which small amounts of transformation products of RDX leach from dried leaves following uptake by poplars. Such behavior for HMX was reported earlier and is reconfirmed here. All three compounds differ substantially in their fate and transport during the leaching process.     

Poly(amidoamine) dendrimer peripherally modified with 4-N,N-dimethylaminoethyleneamino-1,8-naphthalimide as a sensor of metal cations and protons.

A new poly(amidoamine) dendrimer from second generation whose periphery comprises sixteen fluorescent 4-N,N-dimethylaminoethylamino-1,8-naphthalimide units has been synthesized and characterized. In DMF, the dendrimer shows sensitivity to the presence of Cu(2+), Fe(3+) and protons. The changes in the fluorescence intensity of the material are in opposite directions if acids or metals are present. Fluorescence enhancements (FE from 5 to 9 depending on solvent) are recorded when the photoinduced electron transfer (PET) originating from the donating amine to the electron accepting naphthalimide is inhibited by the protonation of the N,N-dimethylamino groups. In the case of Cu(2+) cations, a fluorescence quenching (FQ of 6) is first observed, followed by fluorescence partial restoration. In the Fe(3+) case, the same behaviour is observed with a final FE of 2. The successive complexations of these cations by the dendrimer core and by the external rim of the dendrimer may explain the results.     

Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity

The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.     

Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast

Mating-type switching in the fission yeast Schizosaccharomyces pombe is initiated by a strand-specific imprint located at the mating-type (mat1) locus. We show that the imprint corresponds to a single-strand DNA break (SSB), which is site- but not sequence-specific. We identified three novel cis-acting elements, involved in the formation and stability of the SSB. One of these elements is essential for a replication fork pause next to mat1 and interacts in vivo with the Swi1 protein. Another element is essential for maintaining the SSB during cell cycle progression. These results suggest that the DNA break appears during the S-phase and is actively protected against repair. Consequently, during the following round of replication, a polar double-strand break is formed. We show that when the replication fork encounters the SSB, the leading-strand DNA polymerase is able to synthesize DNA to the edge of the SSB, creating a blunt-ended recombination intermediate.     

15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.     

Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration

Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2‐DE and nanoscale capillary LC/MS/MS. Twenty‐four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration‐regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.