Primary tissue-based proteomic analysis of apoptosis-regulating pathways in malignant pleural mesothelioma (MPM)

PhDMalignant pleural mesothelioma (MPM) is an aggressive and fatal malignancy associated with resistance to chemotherapy and radiotherapy. Programmed cell death or apoptosis underlies the efficacy of cytotoxic anti-cancer modalities, and failure to engage the cellular death machinery in cancer cells accounts for failure of therapy and poor prognosis. Little is understood of the basic apoptosis biology underlying the apoptosis resistant phenotypes of MPM. There is now greater understanding of the basic core apoptosis machinery of the cell, allowing the characterization of anti-apoptosis mechanisms in MPM. Cancer cell apoptosis threshold is ultimately determined by the location and function of proteins. This study explores the expression and significance of several proteins directly or indirectly involved in the apoptosis pathway. Histology samples were used to produce a tissue microarray (TMA) on which immunohistochemistry was employed. Proteins HIF-1α, CD31, Glut-1, CASP3, Survivin, BAX, BAK, Cyt-C, APAF-1 and PARP were analyzed and their protein expression profiles were correlated with patient survival. The findings show that MPM exhibits hypoxia. Increased glucose intake was also demonstrated by overexression of Glut-1. BAX and BAK were shown to be synergistically downregulated, with a direct effect into survival, demonstrating the compromised function of the mitochondria due to increased glucose uptake. Information yielded from this project might allow further exploitation of apoptotic proteins either as targets for new therapeutic agents, or as markers for prognosis

Imaging in pleural mesothelioma: A review of the 11th International Conference of the International Mesothelioma Interest Group

Imaging of malignant pleural mesothelioma (MPM) is essential to the diagnosis, assessment, and monitoring of this disease. The complex morphology and growth pattern of MPM, however, create unique challenges for image acquisition and interpretation. These challenges have captured the attention of investigators around the world, some of whom presented their work at the 2012 International Conference of the International Mesothelioma Interest Group (iMig 2012) in Boston, Massachusetts, USA, September 2012. The measurement of tumor thickness on computed tomography (CT) scans is the current standard of care in the assessment of MPM tumor response to therapy; in this context, variability among observers in the measurement task and in the tumor response classification categories derived from such measurements was reported. Alternate CT-based tumor response criteria, specifically direct measurement of tumor volume change and change in lung volume as a surrogate for tumor response, were presented. Dynamic contrast-enhanced CT has a role in other settings, but investigation into its potential use for imaging mesothelioma tumor perfusion only recently has been initiated. Magnetic resonance imaging (MRI) and positron-emission tomography (PET) are important imaging modalities in MPM and complement the information provided by CT. The pointillism sign in diffusion-weighted MRI was reported as a potential parameter for the classification of pleural lesions as benign or malignant, and PET parameters that measure tumor activity and functional tumor volume were presented as indicators of patient prognosis. Also reported was the use of PET/CT in the management of patients who undergo high-dose radiation therapy. Imaging for MPM impacts everything from initial patient diagnosis to the outcomes of clinical trials; iMig 2012 captured this broad range of imaging applications as investigators exploit technology and implement multidisciplinary approaches toward the benefit of MPM patients. (C) 2013 Elsevier Ireland Ltd. All rights reserved

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 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

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

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

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