Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK

GAS5 long non-coding RNA in malignant pleural mesothelioma

BACKGROUND Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA "sponge". Our aim was to investigate the possible role of the GAS5 in the growth of MPM. METHODS Primary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry. RESULTS GAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression. CONCLUSIONS The observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology

mesothelioma

Background: Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90 % of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA “sponge”. Our aim was to investigate the possible role of the GAS5 in the growth of MPM. Methods: Primary MPM cultures grown in serum-free condition in 3 % oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry

A subset of mesothelioma cells is sensitive to GDC-0980.

<p>(A) M28 and REN monolayers and spheroids were treated with GDC-0980 (1 μM), bortezomib (25 nM), cisplatin (200 μM) plus pemetrexed (10 μM)(C+P) or the combinations for 24 h. Cells with condensed nuclei seen after Hoechst staining were considered apoptotic <i>(* p</i> < <i>0</i>.<i>05</i> compared to the same treatment without GDC-0980; <i>n</i> = 3; mean ± SD). In the spheroids, GDC-0980 had activity when given alone and significantly potentiated chemotherapy in M28 cells but not in REN cells. (B) CaspaseGlo on M28 and REN spheroids treated as previously for 16 h. Caspase 3/7 activation is expressed as relative luminescence units (RLUx10⁵). Again, M28 cells demonstrated sensitivity to GDC-0980 and REN cells demonstrated resistance. <i>(* p</i> < <i>0</i>.<i>05</i> compared to the same treatment without GDC-0980; <i>n</i> = 3; mean ± SD). (C) Six cell lines (M28, SARC, VAMT, REN, JMN, MSTO-211H) grown as spheroids were treated with GDC-0980 (1 μM), bortezomib (25 nM) or the combination for 24 h. Three cell lines showed a response to GDC-0980 (<i>sensitive</i>) and 3 showed no response (<i>resistant</i>). <i>(* p</i> < <i>0</i>.<i>05</i> compared to the same treatment without GDC-0980; <i>n</i> = 3; mean ± SD) (D) Caspase 3/7 activation in spheroids treated similarly for 16 h, measured by CaspaseGlo, confirmed the differences between the sensitive and resistant spheroids. <i>(* p</i> < <i>0</i>.<i>05</i> different from the response to chemotherapy alone; <i>n</i> = 3; mean ± SD) (E) M28 spheroids were treated with higher concentrations of GDC-0980 (1,10 and 20 μM) for 24 h and caspase 3/7 activity was measured by CaspaseGlo. Higher concentrations of GDC-0980 did not increase its efficacy.</p

GDC-0980 efficacy does not correlate with Akt/mTOR activation in tumor fragment spheroids.

<p>(A) M28 and REN cells grown as monolayers (<i>m</i>) or spheroids (<i>s</i>) were treated with GDC-0980 (1 μM) for 6 h and studied by immunoblotting for phosphorylated Akt, S6K and ERK. Mesothelioma cells down-regulated the Akt/mTOR pathway when grown as 3D multicellular spheroids, as we have previously shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134825#pone.0134825.ref001" target="_blank">1</a>]. GDC-0980 inhibited P-Akt and P-S6K in both monolayers and spheroids. P-ERK, used as a control, was not affected by GDC-0980. (B) GDC-<i>sensitive</i> (<i>S</i>) and-<i>resistant</i> (<i>R</i>) spheroids were treated with GDC-0980 (1 μM) for 6 h and analyzed by immunoblotting for P-Akt, P-S6K and P-ERK. Multicellular spheroids sensitive to GDC-0980 had higher P-Akt than resistant ones. (<b>C</b>) The 21 tumor fragment spheroids with a known response to GDC-0980 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134825#pone.0134825.g002" target="_blank">Fig 2</a>) were studied for expression of P-Akt by immunohistochemistry. In the tumor fragments, in contrast to the multicellular spheroids, response to GDC-0980 did not correlate with P-Akt levels <i>(p = 0</i>.<i>6713</i> sensitive vs resistant, n = 11 for the sensitive group, n = 10 for the resistant group).</p

Inhibition of autophagy potentiates the response to chemotherapy only in the spheroids sensitive to GDC-0980.

<p>(A) M28 and REN spheroids were transfected with ATG5 (left panels) and ATG7 siRNA (right panels) and treated with GDC-0980 (1 μM), bortezomib (25 nM), ammonium chloride (NH₄⁺, 10 mM) or the combination for 6 h. Both ATG5 and ATG7 siRNA effectively reduced their respective protein levels to less than 10% of the original levels. However, in M28 and REN spheroids, ATG5 siRNA failed to reduce LC3-II levels, showing that autophagy was not inhibited. Instead, in both M28 and REN spheroids, ATG7 siRNA decreased LC3-II levels, showing that it was effective in blocking autophagy. Of note, bortezomib did not affect the autophagic flux of either spheroid. (B) The band intensities in 3 separate immunoblots were measured by densitometry and the ratios of LC3 II to tubulin values (LC3 vs tubulin) are shown. Statistical significance was calculated by ANOVA with Tukey’s test (<b>*</b> scrambled siRNA vs ATG5 or 7 siRNA; <i>p < 0</i>.<i>05</i>; n = 3; mean ± SD). (C) M28 and REN spheroids, grown from cells transfected with ATG5 or ATG7 siRNA, were treated with GDC-0980 (1 μM), bortezomib (25 nM)(BZ) or cisplatin (200 μM) plus pemetrexed (10 μM)(C+P) for 16 h. Apoptosis was measured by a CaspaseGlo assay. The response to GDC-0980 and to chemotherapy was potentiated by ATG7 siRNA, only in M28 spheroids. <i>(* p</i> < <i>0</i>.<i>05</i> compared to scrambled control siRNA; <i>n</i> = 3; mean ± SD).</p

ATG13 puncta are present only in the spheroids sensitive to GDC-0980.

<p>(A) Multicellular spheroids found to be <i>sensitive</i> (M28, SARC, VAMT) or <i>resistant</i> (REN, JMN, MSTO-211H) to GDC-0980 were immunostained for ATG13. Only the spheroids sensitive to GDC-0980 displayed ATG13 puncta. (B) Tumor fragment spheroids that were found to be sensitive or resistant to GDC-0980 were stained for ATG13. All the tumor fragment spheroids sensitive to GDC-0980 had more than 10% cells positive for ATG13 puncta, whereas the resistant group showed little to no ATG13 staining. <i>(* p</i> < <i>0</i>.<i>05</i> sensitive vs resistant; n = 11 for the sensitive group, n = 10 for the resistant group; mean ± SEM). Panels are representative images of tumor fragment spheroids stained for pan-cytokeratin (<i>red</i>) and ATG13 (<i>green</i>). An enlarged panel is also provided showing ATG13 puncta in a sensitive tumor fragment spheroid, as indicated by arrows (<i>scale bar for both panels 10</i>μ<i>m</i>).</p

GDC-0980 efficacy is caspase-dependent but does not require Bim or Bid.

<p>(A) M28 spheroids were treated with GDC-0980 (1 μM), bortezomib (25 nM) or the combination with or without the pan-caspase inhibitor zVAD-fmk (20 μM) for 8, 16 and 24 h. Apoptosis was measured by counting nuclear condensation of disaggregated cells stained with Hoechst. GDC-0980 activity was completely abolished by zVAD-fmk at all time points. <i>(* p</i> < <i>0</i>.<i>05</i> different from GDC-0980 plus bortezomib; <i>n</i> = 3; mean ± SD). (B) M28 spheroids, transfected with a control scrambled siRNA (10 nM) or with a combination of Bim siRNA and Bid siRNA (10 nM), were treated with GDC-0980 (1 μM), bortezomib (25 nM), or the combination with or without zVAD-fmk and apoptosis measured. SAHA (5 μM) was used as positive control for a requirement for Bim/Bid, as we have previously shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134825#pone.0134825.ref028" target="_blank">28</a>]. Efficacy of knockdown was measured by immunoblot (see insert). Response to GDC-0980 was not affected by Bim/Bid siRNA but was abolished by zVAD-fmk. The response to SAHA was significantly reduced by the Bim/Bid knockdown, confirming effective knockdown. <i>(* p</i> < <i>0</i>.<i>05</i> different from scrambled control siRNA; <i>n</i> = 3; mean ± SD). In sum, the apoptotic response to GDC-0980 does not require Bim and Bid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134825#pone.0134825.ref001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134825#pone.0134825.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134825#pone.0134825.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134825#pone.0134825.ref055" target="_blank">55</a>].</p