PSMB7 is associated with anthracycline resistance and is a prognostic biomarker in breast cancer

BACKGROUND: To date individual markers have failed to correctly predict resistance against anticancer agents in breast cancer. We used gene expression patterns attributable to chemotherapy-resistant cells to detect potential new biomarkers related to anthracycline resistance. One of the genes, PSMB7, was selected for further functional studies and clinical validation. METHODS: We contrasted the expression profiles of four pairs of different human tumour cell lines and of their counterparts resistant to doxorubicin. Observed overexpression of PSMB7 in resistant cell lines was validated by immunohistochemistry. To examine its function in chemoresistance, we silenced the gene by RNA interference (RNAi) in doxorubicin-resistant MCF-7 breast cancer cells, then cell vitality was measured after doxorubicin treatment. Microarray gene expression from GEO raw microarray samples with available progression-free survival data was downloaded, and expression of PSMB7 was used for grouping samples. RESULTS: After doxorubicin treatment, 79.8+/-13.3% of resistant cells survived. Silencing of PSMB7 in resistant cells decreased survival to 31.8+/-6.4% (P>0.001). A similar effect was observed after paclitaxel treatment. In 1592 microarray samples, the patients with high PSMB7 expression had a significantly shorter survival than the patients with low expression (P<0.001). CONCLUSION: Our findings suggest that high PSMB7 expression is an unfavourable prognostic marker in breast cancer

miRpower: a web-tool to validate survival-associated miRNAs utilizing expression data from 2178 breast cancer patients

PURPOSE: The proper validation of prognostic biomarkers is an important clinical issue in breast cancer research. MicroRNAs (miRNAs) have emerged as a new class of promising breast cancer biomarkers. In the present work, we developed an integrated online bioinformatic tool to validate the prognostic relevance of miRNAs in breast cancer. METHODS: A database was set up by searching the GEO, EGA, TCGA, and PubMed repositories to identify datasets with published miRNA expression and clinical data. Kaplan-Meier survival analysis was performed to validate the prognostic value of a set of 41 previously published survival-associated miRNAs. RESULTS: All together 2178 samples from four independent datasets were integrated into the system including the expression of 1052 distinct human miRNAs. In addition, the web-tool allows for the selection of patients, which can be filtered by receptors status, lymph node involvement, histological grade, and treatments. The complete analysis tool can be accessed online at: www.kmplot.com/mirpower . We used this tool to analyze a large number of deregulated miRNAs associated with breast cancer features and outcome, and confirmed the prognostic value of 26 miRNAs. A significant correlation in three out of four datasets was validated only for miR-29c and miR-101. CONCLUSIONS: In summary, we established an integrated platform capable to mine all available miRNA data to perform a survival analysis for the identification and validation of prognostic miRNA markers in breast cancer

DLK-1, SEK-3 and PMK-3 Are Required for the Life Extension Induced by Mitochondrial Bioenergetic Disruption in <i>C</i>. <i>elegans</i>

<div><p>Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode <i>C</i>. <i>elegans</i> not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPR^mt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene <i>Ptbb-6</i>::<i>GFP</i>. Inhibition of known mitochondrial retrograde responses does not alter induction of <i>Ptbb-6</i>::<i>GFP</i>, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate <i>Ptbb-6</i>::<i>GFP</i>, we find that <i>dlk-1</i>, <i>sek-3</i> and <i>pmk-3</i> are all required for their life extension.</p></div

Role of <i>pmk-3</i> in Mit mutant life extension.

<p>(<b>A-D</b>) <i>pmk-3(ok169)</i> worms showed significantly attenuated life extension upon RNAi knockdown of specific mitochondrial ETC subunits. (<b>E-H</b>) <i>isp-1(qm150)</i> and <i>tpk-1(qm162)</i>, but not <i>nuo-6(qm200)</i> or <i>clk-1(qm30)</i> mitochondrial ETC mutants display significantly attenuated life extension following RNAi-mediated removal of <i>pmk-3</i>. For these four panels, lifespan curves represent averages of two or more independent experiments. Significance values, N, and lifespan statistics are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.s015" target="_blank">S4 Table</a>. Raw lifespan data is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.s016" target="_blank">S5 Table</a>.</p

<i>Ptbb-6</i>::<i>GFP</i> reporter expression defines a UPR^mt independent pathway.

<p>(<b>A</b>) RNAi knockdown of <i>atfs-1</i> blocks <i>Phsp-6</i>::<i>GFP</i> expression, as reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.ref043" target="_blank">43</a>], but dramatically further upregulates <i>Ptbb-6</i>::<i>GFP</i> in both <i>isp-1(qm150)</i> and <i>nuo-6(qm200)</i> worms. Surprisingly, <i>atfs-1</i> RNAi also turned off <i>Pgst-4</i>::<i>GFP</i>. RNAi knockdown of <i>skn-1</i> in both <i>isp-1(qm150)</i> and <i>nuo-6(qm200)</i> worms (data for latter worms is also provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.s007" target="_blank">S7 Fig</a>), turns off <i>Pgst-4</i>::<i>GFP</i>, as reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.ref059" target="_blank">59</a>], but has no effect on <i>Ptbb-6</i>::<i>GFP</i> (and <i>Phsp-6</i>::<i>GFP)</i> expression. (<b>B</b>) Upregulation of <i>Ptbb-6</i>::<i>GFP</i> following <i>atfs-1</i> removal is only observed in the context of ETC dysfunction. (<b>C</b>) Quantitative of <i>atfs-1</i> mRNA in worms of panel (B). Bars represent mean (+/- SD); <i>n</i> = 3 biological replicates/condition. Asterisks indicate significant knockdown of <i>atfs-1</i> mRNA on <i>atfs-1</i> RNAi relative to vector (Student’s t-test, *p<0.001, **p<0.0001).</p

RNAi-mediated inhibition of <i>tbb-6</i> mildly inhibits the life extension of <i>isp-1(qm150)</i> Mit mutants—potentially by mediating voltage-dependent anion channel (VDAC) activity.

<p>(<b>A)</b> Survival analysis of <i>isp-1(qm150)</i> and wild type (N2) worms cultured on RNAi to <i>tbb-6</i> or vector control (pL4440). The lifespan of <i>isp-1(qm150)</i> mutants is reduced by ~7% following knockdown of <i>tbb-6</i>. (Combined data from replicate experiments, Log rank test <i>p</i> < 0.003, N = 126–206 worms/condition). Full lifespan statistics in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.s015" target="_blank">S4 Table</a>. (<b>B</b>) C-termini of β-tubulins from various species. Non-<i>C</i>. <i>elegans</i> data derived from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.g006" target="_blank">Fig 6A</a> of Rostovtseva and colleagues [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.ref101" target="_blank">101</a>]). (<b>C</b>) Removal of either of the two C/EBP-like motifs in the <i>tbb-6</i> promoter of <i>Ptbb-6</i>::<i>GFP</i> abrogates GFP reporter expression. Transgenic lines contain mCherry under the control of the wild-type <i>tbb-6</i> promoter as an internal control.</p

<i>Ptbb-6</i>::<i>GFP</i> expression requires a MAPK signal cascade.

<p>(<b>A</b>) RNAi-mediated disruption of the MAP3K/MAP2K/MAPK pathway defined by DLK-1 → SEK-3 → PMK-3 blocks induction of <i>Ptbb-6</i>::<i>GFP</i> in <i>isp-1(qm150)</i> worms but not <i>Pgst-4</i>::<i>GFP</i> nor <i>Phsp-6</i>::<i>GFP</i> reporter expression. Graph provides quantification of reporter expression level, normalized to vector-control RNAi (Mean+/-SD, <i>n =</i> 12–18 worms/ RNAi treatment). Asterisks indicate significant difference relative to vector (one-way ANOVA and <i>ad hoc</i> using Dunnett’s Multiple Comparisons Test, *p<0.05, **p<0.01, ***p<0.001). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.s009" target="_blank">S9 Fig</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.s014" target="_blank">S3 Table</a>. (<b>B</b>) <i>pmk-3(ok169)</i> null mutants show the expected reduction in size upon RNAi knockdown of mitochondrial respiratory subunits, but are incapable of inducing <i>Ptbb-6</i>::<i>GFP</i>. (<b>C</b>) MKK-4 is not required for <i>Ptbb-6</i>::<i>GFP</i> induction following mitochondrial ETC disruption by <i>isp-1</i> RNAi, unlike SEK-3. (<b>D</b>) Neuron-specific expression of a constitutively active form of DLK-1 acts cell autonomously to activate <i>Ptbb-6</i>::<i>GFP</i> expression. There is no induction of <i>Ptbb-6</i>::<i>GFP</i> in intestinal or other cells. (Arrows mark ventral nerve cord).</p

Evidence for a novel signaling pathway activated subsequent to mitochondrial disruption.

<p>(<b>A</b>) Among the ten most highly upregulated genes activated following mitochondrial disruption by <i>spg-7</i> RNAi, <i>tbb-6</i> alone does not require <i>atfs-1</i> for its induction (microarray data from GEO dataset GSE38196). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.s012" target="_blank">S1 Table</a>. (<b>B</b>) 40 of the 148 <i>atfs-1</i> independent genes activated following <i>spg-7</i> disruption contain a predicted EOR-1 binding motif (shown in LOGO form aligned against the consensus EOR-1 site which was identified through the <i>C</i>. <i>elegans</i> ModENCODE project (<i>top panel</i>)). (<b>C</b>) 42 of the 148 <i>atfs-1</i> independent genes activated following <i>spg-7</i> disruption contain a C/EBP-like promoter motif (shown in LOGO form aligned against the promoter motif bound by human C/EBPβ (<i>top panel</i>)). (<b>D</b>) Venn diagram illustrating the degree of overlap between groups of <i>atfs-1</i> independent genes that contain C/EBPβ -like, EOR-1 or DAF-16 promoter elements. (<b>E</b>) Promoter region of <i>tbb-6</i>: Sites A and B match the human C/EBPβ consensus motif shown in panel (C). ChiP-Seq data from the <i>C</i>. <i>elegans</i> ModENCODE project [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.ref052" target="_blank">52</a>], reveals a functional DAF-16 binding site, as well as a functional PHA-4 binding site [Stv. L1(rep 2)–starved L1 larvae, 2^nd replicate sample set, L4/YA—larval stage 4/young adult].</p