Expression profiling stratifies mesothelioma tumors and signifies deregulation of spindle checkpoint pathway and microtubule network with therapeutic implications

Abstract

Background: Malignant pleural mesothelioma (MPM) is a lethal neoplasm exhibiting resistance to most treatment regimens and requires effective therapeutic options. Though an effective strategy in many cancer, targeted therapy is relatively unexplored in MPM because the therapeutically important oncogenic pathways and networks in MPM are largely unknown.

Materials and methods: We carried out gene expression microarray profiling of 53 surgically resected MPMs tumors along with paired normal tissue. We also carried out whole transcriptomic sequence (RNA-seq) analysis on eight tumor specimens. Taqman-based quantitative Reverse-transcription polymerase chain reaction (qRT-PCR), western analysis and immunohistochemistry (IHC) analysis of mitotic arrest deficient-like 1 (MAD2L1) was carried out on tissue specimens. Cell viability assays of MPM cell lines were carried out to assess sensitivity to specific small molecule inhibitors.

Results: Bioinformatics analysis of the microarray data followed by pathway analysis revealed that the mitotic spindle assembly checkpoint (MSAC) pathway was most significantly altered in MPM tumors with upregulation of 18 component genes, including MAD2L1 gene. We validated the microarray data for MAD2L1 expression using quantitative qRT-PCR and western blot analysis on tissue lysates. Additionally, we analyzed expression of the MAD2L1 protein by IHC using an independent tissue microarray set of 80 MPM tissue samples. Robust clustering of gene expression data revealed three novel subgroups of tumors, with unique expression profiles, and showed differential expression of MSAC pathway genes. Network analysis of the microarray data showed the cytoskeleton/spindle microtubules network was the second-most significantly affected network. We also demonstrate that a nontaxane small molecule inhibitor, epothilone B, targeting the microtubules have great efficacy in decreasing viability of 14 MPM cell lines.

Conclusions: Overall, our findings show that MPM tumors have significant deregulation of the MSAC pathway and the microtubule network, it can be classified into three novel molecular subgroups of potential therapeutic importance and epothilone B is a promising therapeutic agent for MPM.

Keywords: MAD2L1; epothilone B; mesothelioma; microarray; network; therapeutics.

Figure 1.

Robust cluster analysis identifies three molecular subgroups of mesothelioma tumors. In a process repeated 1000 times, a uniform number of probesets (500–2000) are randomly selected from a set selected at the FDR level of 0.01 (a total of 20 000 probesets). During each process, whenever any two samples are grouped together, it is recorded to generate the consensus matrix which denotes the average time any two samples are grouped together in each robust clusters. The consensus matrix is used as the similarity matrix to define the final clusters. The intensity bar below denotes the values of color coding used in the cluster map.

Figure 2.

Heat map using ANOVA identifies probesets/genes different among the three molecular groups. There are actually two different expression groups within subgroup 3—one called subgroup 3 while the other called minor subgroup 3. These three subgroups correlate partially with histology but did not correlate with the run date and therefore not accounted by any batch effect. Mitotic spindle assembly checkpoint (MSAC) genes are differentially expressed with highest level of expression in subgroup 3 and minor subgroup 3 while other expression groups have upregulation of specific pathways.

Figure 3.

Transcript and protein analysis of MAD2L1 in tumors and cell lines. (A) qPCR analysis of MAD2L1 message in MPM tumor samples compared with normal paired samples. Twenty-three paired samples were analyzed for MAD2L1 expression using qPCR assays with GAPDH as normalizing endogenous controls and 2^−ΔCt were plotted on a log₁₀ scale to compare between the sets. The box plot shows expression in the tumors was significantly higher compared with paired normal control samples with a 3-fold difference in the means (***P = 0.0036). The ‘+’ marks the mean for the box plots. (B) MAD2L1 protein expression in tumor tissue. Western blots of tumor (N = 80 cases) and normal (N = 54 cases) were probed for MAD2L1- and β-actin-specific antibodies. After scanning, quantizing and normalizing the autoradiograms, the tumors show 2.7-fold increase in mean value of MAD2L1 protein, compared with β-actin (***P < 0.0001). The normalized intensity values were plotted on the Y-axis on a log₁₀ scale. The ‘+’ marks the mean for the box plots. (C) Western blot analysis of three MPM cell lines. Protein lysates isolated from different cellular compartments were probed with MAD2L1- and control biomarkers-specific antibodies. The nuclear (Nuc), cytoplasmic (Cyto) and total (Total) levels of MAD2L1 protein are depicted in the blot. Nuclear lamin A and Nuclear lamin C serve as controls for presence of nuclear proteins while β-actin serves as loading control.

Figure 4.

MAD2L1 protein expression in mesothelioma tumors by IHC. (A) Representative histotypes of epithelioid type (a, ×200; b, ×400; c, ×200), and sarcomatoid type (d, ×200; e, ×400; f, ×200). Cases depicted in pictures a, b, d and e show positive nuclear moderate and strong positive nuclear signal of MAD2L1 protein in malignant tumor MPM cells. Cases depicted in pictures c and f show negative staining in malignant cells. (B) Kaplan–Meier showing overall survival of all MPMs by nuclear MAD2L1 expression using the median expression (44.0) as cutoff. (C) Kaplan–Meier showing overall survival of epitheloid MPMs by nuclear MAD2L1 expression using the median (43.3) expression as cutoff.