Triple-negative breast cancer (TNBC) is defined by a lack of expression of estrogen, progesterone, and HER2 receptors, and genetically most of them fall into the basal subgroup of breast cancer. The important issue of TNBC is poorer clinical outcome and absence of effective targeted therapy. In this study, we sought to identify DNA copy number alterations and expression of relevant genes characteristic of TNBC to discover potential therapeutic targets. Frozen tissues from 114 breast cancers were analyzed using high-resolution array comparative genomic hybridization. The classification into subtype was determined by estrogen and progesterone receptor expression, and by the presence or absence of gain on the ERBB2 containing clone. The ACE algorithm was used for calling gain and loss of clones. Twenty-eight cases (25%) were classified as TNBC. Recurrent gains (> or =25%) unique to TNBC were 9p24-p21, 10p15-p13, 12p13, 13q31-q34, 18q12, 18q21-q23, and 21q22. Two published gene expression array data sets comparing basal subtype versus other subtype breast cancers were used for searching candidate genes. Of the genes upregulated in the basal subtype, 45 of 686 genes in one data set and 59 of 1,428 in the second data set were found to be located in the gained regions. Of these candidate genes, gain of NFIB (9p24.1) was specific for TNBC in a validation set by real-time PCR. In conclusion, we have identified recurrently gained regions characteristic of TNBC, and found that NFIB copy number and expression is increased in TNBC across the data sets. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

We have previously determined that sustained phospholipase D (PLD) activation is associated with differentiation induction in primary mouse epidermal keratinocytes. We therefore investigated the effect of two bacterial PLD on keratinocyte proliferation and differentiation. We found that Streptomyces sp. PLD was much less potent at inhibiting proliferation than S. chromofuscus PLD, with a half-maximal inhibitory concentration of 0.05 versus less than 0.001 IU per ml for S. chromofuscus PLD. Similarly, S. chromofuscus PLD stimulated transglutaminase activity more effectively and potently than S. sp. PLD. When we examined the formation of products by the two PLD, we found that the S. sp. PLD showed higher activity at all concentrations. Whereas the PLD from S. sp. is relatively inactive on sphingomyelin, S. chromofuscus PLD is known to hydrolyze both glycerophospholipids and sphingomyelin. Based on recent data indicating a role for ceramide in regulating cell growth and differentiation, we hypothesized that the ability of S. chromofuscus PLD to hydrolyze sphingomyelin might underlie its greater potency. Therefore, we examined the effect of exogenous sphingomyelinase and synthetic ceramides on DNA synthesis. We found that sphingomyelinase exhibited a potent concentration-dependent effect on [3H]thymidine incorporation, much like S. chromofuscus PLD. Synthetic cell-permeable ceramides (C6- and C2-ceramide) also concentration dependently inhibited DNA synthesis, with a half-maximal inhibitory concentration of approximately 12 microM. Finally, we obtained evidence suggesting that ceramide is generated in response to a physiologically relevant agent, because tumor necrosis factor-alpha, a known effector of sphingomyelin turnover in other systems and a cytokine that is produced and released by keratinocytes, increased ceramide levels in primary epidermal keratinocytes.

Much data in the literature suggest a role for protein kinase C (PKC) in regulating keratinocyte proliferation and differentiation. Nevertheless, the exact role of this family of isoenzymes is unclear, since PKC agonists (e.g., phorbol esters) are known to stimulate expression of both proliferative and differentiative markers in keratinocytes. Similarly, PKC inhibitors have been demonstrated both to inhibit [2-[1-3(aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide, acetate (Ro 31-7549) and 3- [1-[3-(amidinothio)propyl-1H-indol-3-(1-methyl-1H-indol-3yl) maleimide (Ro 31-8220)] and to induce (staurosporine) keratinocyte differentiation. In this study, we examined the role of the PKC inhibitor, Gö decke 6976 (Gö 6976) [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo (3,4-c)-carbazole], on keratinocyte proliferation, as measured by DNA synthesis, and differentiation, as monitored by transglutaminase activity. This compound is reported to be selective for the conventional PKC isoforms, of which keratinocytes express only PKC␣, and for protein kinase D (PKD; also known as PKC). We report that Gö 6976 stimulated transglutaminase activity. Consistent with this effect, Gö 6976 also potently inhibited [3 H]thymidine incorporation (a half-maximal inhibitory concentration of ϳ0.1 M). In addition, Gö 6976 (1 M) was able to enhance the stimulation of transglutaminase activity by 1,25-dihydroxyvitamin D 3 but had no effect on D 3 -induced expression of keratin-1. Conversely, Gö 6983 [2-[1-(3-dimethylaminopropy)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], a similar compound that also selectively inhibits conventional PKC␣, but not PKD, had little or no effect on DNA synthesis or transglutaminase activity (up to 1 M). The effect of Gö 6976 was not due to cytotoxicity as its effect on thymidine incorporation was largely reversible, and its stimulation of transglutaminase activity could be inhibited by another general PKC inhibitor, bisindolylmaleimide I. Therefore, our results suggest a proproliferative, antidifferentiative role for PKD in epidermal maturation.The epidermis is composed primarily of epidermal keratinocytes, which continuously proliferate and differentiate to maintain this important tissue. Keratinocyte differentiation is characterized by a spatially and temporally regulated program of gene and protein expression, which ultimately results in terminal differentiation and cell death. This program of differentiation is essential for the function of the epidermis as a barrier to water loss, microbial invasion, and mechanical stress. Despite the importance of keratinocyte differentiation to epidermal structure, the signaling pathways that regulate this process are not well understood. Numerous data in the literature indicate a role for PKC in keratinocyte differentiation; however, the exact role of this enzyme is at present unclear (reviewed in Bollag and Bollag, 2001). Thus, PKC-activating phorbol esters elicit events associated paradoxically both with differentiati...

Ambient particulate matter (PM) induces adverse health effects through the ability of pro-oxidative chemicals to induce the production of oxygen radicals and oxidant injury. Utilizing a proteomics strategy involving 2-D DIGE, immunoblotting, and real-time PCR, we demonstrate that organic diesel exhaust particle (DEP) chemicals induce an unfolding protein response (UPR) and proinflammatory effects in the human bronchial epithelial cell line, BEAS-2B. DIGE and MS showed the induction of at least 14 proteins, among which heat shock protein 70 (HSP70), HSP40, TPR2, and T-complex protein 1 (zeta-subunit) are known to play a role in the UPR. Demonstrating increased HSP70 mRNA expression and nuclear translocation of HSF1, the key transcription factor responsible for HSP expression, further strengthened this notion. Immunoblotting demonstrated increased expression of ATF4, an ER stress-associated transcriptional enhancer responsible for differential protein translation under conditions of ER stress. Finally, the DEP extract induced the expression of IL-6 and IL-8 in the culture supernatant. The role of oxidative stress was demonstrated further by response subtraction in the presence of the thiol antioxidant, N-acetyl cysteine. Our data suggest that pro-oxidative DEP chemicals induce protein unfolding/misfolding that lead to UPR and proinflammatory effects in a cell type that is targeted by PM in the lung.

Previously, we reported that DNA copy number of NFIB is amplified in triple negative breast cancer (TNBC; estrogen(−)/progesterone(−)/HER2(−)). The transcriptional activator NFIB plays wide roles in regulation of gene transcription, cell proliferation, viral DNA replication and development. But the functional role of NFIB in breast cancer cell has not been elucidated. In order to understand the role of upregulated NFIB in breast cancers, we examined mRNA and protein level of NFIB in 20 breast cancer tissues. In ER negative breast cancer subtype, expression of NFIB is up-regulated and correlated with Ki67 expression. And intracellular localization of NFIB is restricted in nucleus. In ER negative cell line, knock-down of NFIB leads to reduced cell proliferation by cell cycle arrest. Cell cycle arrest is accompanied by S phase arrest and alteration of S phase regulating protein expression. Take together, transcription activator of NFIB regulates cancer cell proliferation in ER negative breast cancer and it may act as a therapeutic marker. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C26.