signature=69a22092d66a85285836ed87476f1378,Lung Cancer Signature Biomarkers: tissue specific semanti...

DDD based prioritisation of lung cancer genes

In order to find the lung tissue specific differentially expressed genes, two Unigene pools (A and B) were constructed (See Additional file 1). For analysis, in the DDD1, we employed the UniGene pool (A) representing 39 human normal tissues excluding normal lung tissue and UniGene pool (B) representing 11 counterpart lung normal tissues were employed for analysis (Table 1). Similarly, in DDD2, UniGene pool (A) representing 8 human lung tumours and UniGene pool (B) representing 11 counterpart lung normal tissues were employed (Table 1). The fold change of normal lung (DDD1) and lung carcinoma candidate genes (DDD2) were calculated based on transcript frequency values. The candidate genes with an expression of at least 2-fold difference were taken into analysis. In DDD1, amongst the total of 519 differentially expressed genes 268 genes were up-regulated (≥2-fold) and 234 genes were down-regulated (≥2-fold). In DDD2, amongst the total of 203 differentially expressed candidate genes, 147 genes (≥2-fold) including 33 unknown were up-regulated (≥2-fold) and 55 genes were down-regulated (≥2-fold). Comparison of DDD1 with DDD2 has revealed that in total 76 genes from DDD1 were differentially expressed in DDD2 (See Additional file 2). From the literature survey, amongst the 76 genes, 18 of them were found to be commonly expressed in all types of cancerous conditions (See Additional file 3) [16]. Excluding these 18 from the 76, the remaining 58 genes were predicted as the lung tissue specific tumour genes (See Additional file 2). The molecular functions of these 58 genes were found to be involved in broad range of cellular functions with majority of the genes playing many different roles like structural, extracellular and intracellular functions. This subtractive approach eliminated most of the commonly expressing genes; for example, housekeeping genes. This approach has also helped to eliminate genes expressing in more than 10 cancerous conditions (See Additional file 4).

Table 1

Different tissue specific Unigene libraries employed in DDD

Prediction of Lung tissue specific tumour genes by Semantic similarity score based clustering

To identify lung tissue specific clusters from the 202 genes from DDD2 cancerous condition, firstly they were subjected to similarity clustering analysis using the 47 lung tissue specific genes from TiSGeD (See Additional file 5). Before the semantic similarity clustering analysis, the Unigene ID were converted into Entrez ID. During this process, the 202 genes of DDD2 reduced to 145 and the 47 lung tissue specific genes of TiSGeD were reduced to 28 due to gene duplication. Using GOSemSim package, the similarity correlation matrix was constructed between the 145 predicted lung specific differentially expressed cancer genes from DDD2 and 28 genes from TiSGeD. The differential expression levels of these clustered genes were depicted in the form of a Heat Map (Figure 1). The similarity correlation matrix produced seven gene clusters at 95% confidence level, using the pvclust program (Figure 2). The clusters 1–4 have 14 genes and the clusters 5, 6 and 7 have 36, 74 and 14 genes respectively.

Figure 1

Go semantic similarity score between the set of normal lung tissue specific genes from TiSGeD (28-horizontal, x-axis) and the differentially expressed lung cancer genes from DDD2 (145-vertical, y-axis). The intensity of the color corresponds to the magnitude of the similarity. Red represents low semantic similarity below the median level whereas the green represents high semantic similarity above the median level.

Figure 2

Average correlation distances with hierarchical clustering based on GO semantic similarity score matrix calculated between normal lung tissue specific genes from TiSGed and differentially expressed lung cancer gene from DDD2. Values in red represent AU (Approximately unbiased) p-value and green represents BP (Bootstrap probability) Clusters with AU larger than 95% are highlighted by red rectangle boxes. AU p-value, which is computed by multiscale bootstrap resampling, is a better approximation to unbiased p-value than BP value computer by normal bootstrap resampling.

In the ID conversions from Unigene to Entrez, the 58 lung tissue specific tumour genes were reduced to 38 genes (Table 2). These 38 genes were matched with the 7 clusters. This38 genes formed four panels with the corresponding cluster 4, 5, 6 and 7 respectively. The panels 1–4 contained 2, 9, 21 and 6 genes respectively. This leads to identification of the lung tissue specific clusters of the normal lung tissue specific genes differentially regulated in lung cancer condition.

Table 2

Lung cancer signature biomarker clusters

We then analysed the functional significance of each panel as given below.

Analysis of Cluster 4 / Panel 1

The cluster 4 had two-lung cancer related genes ubiquitin thiolesterase (UCHL1) and Lactotransferrin (LTF). In the normal lung (DDD1 data), UCHL1 was down-regulated and LTF was up-regulated (Table 2). This was reversed during the lung cancer condition where UCHL1 up-regulated and the LTF highly down-regulated (Table 2). These two proteins were found to be important in the cancer progression. UCH-L1 up-regulation promoted prostate cancer metastasis through epithelial-to-mesenchymal transition (EMT) induction and LTF expression decreased in lung prostate cancer progression [17, 18]. Both of them were co-expressed in almost six different lung adenocarcinoma cell lines, as evident by mSigDB. This suggested that UCH-L1 and LTF could be novel diagnostic and therapeutic targets for lung cancer metastasis diagnostic markers.

Analysis of Cluster 5 / Panel 2

The cluster 5 was playing the common functional role of immune response and complement activation. The down-regulated RPSA, RPL9, TMSB4X and TUBA1B in normal lung (DDD1) were significantly up-regulated in lung cancer (DDD2) (Table 1). The analysis resulted that all these up regulated genes played the role of tumour cell resistance to the anti-cancer agents. In gastric cancers, the up-regulation of RPSA/LRP contributed to drug resistance via hypoxia-inducible-factor dependent mechanism [19]. Similarly, there was a link between the TMSB4X and TUBA1B and the anti-cancer drug resistance to the drug Paclitaxel (PTX) observed in the cervical and breast/ovarian cancers respectively [20, 21].

In this cluster, NT5C2, API5, CPN, PRKAR1A and COPB1 were fully down-regulated in lung cancer (Table 1). The down regulation of NT5C3 altered the tumour cell sensitivity to cytidine based anti-cancer drugs [22]. The anti-apoptosis gene API5 down-regulation linked to increase in the survival and resistance cancer cells to chemotherapy [23]. To our knowledge, the major copper carrying protein CPN (ceruloplasmin) down regulation link to chemotherapy/drug resistance is not yet studied. But increased level of copper in lewis lung carcinoma cells were related with the development of multi drug resistance [24]. The PRKAR1A down-regulation also linked to multidrug-resistant (MDR) in colon carcinoma cells [25]. The COPB1 was an essential component for the coatomer formation [26]. These coatomers were involved in the drug trafficking pathways and endocytic drug delivery [27]. So, it was expected that the down-regulation of COPB1 might have a role in the chemotherapy which needs to be taken up and studied. We are surprised to find that all these results suggest that the cluster 5 functionally represents a panel of chemotherapy/drug resistance related lung cancer biomarkers.

Analysis of Cluster 6 / Panel 3

In cluster 6, the upregulated FTL (65 fold in our study) and ALDOA (7 fold in our study) were regulated by hypoxia inducible factor (HIF) during lung cancer [28–31]. The COL1A1 (23 fold in our study) and GAPDH (11 fold in our study) were regulated by hypoxia [32–34]. IGKC (8 fold in our study) up-regulated in lung cancer patients but no literature data was available for its interaction either with HIF or hypoxia [35]. The HIF, TGM2, CSNK1A1, CSNK2A1, CTNNA1, NAMPT)/Visfatin, TNFRSF1A, ETS1 and SRC-1 were down-regulated and proposed as the biomarkers for lung cancer. We found all of them to be interacting with the HIF in cancerous condition [36–45]. The down- regulated FN1 and APLP2 showed hypoxia dependent differential regulation [46–48]. The DMBT1/SAG interacted HIF-1 was a kind of feedback loop in response to hypoxia. The hypoxia induced HIF-1 to transactivate SAG and the induced SAG then promoted HIF-1alpha ubiquitination and degradation [49]. The FBJ/c-Jun/AP-1 interacted with HIF during hypoxia that controlled the transcriptional regulation of the Cyr61 gene in retinal vascular endothelial cells [50]. The role of AIB1/SRC-3/NCoA during hypoxia condition were exhibited by controlling the expression levels of HIF induced erythropoietin (EPO) gene during hypoxia [42].

However, in this cluster, the AZIN1 and TICAM2 were down-regulated and were lacking direct experimental evidence to support their regulation with HIF or hypoxia during cancer. The following literature analysis suggests their possible regulations either with HIF or hypoxia. The AZIN1 was an inhibitor for the antizyme and both were highly regulated in human cancers and antizyme induced HIF, during increased cellular redox potential [51–53]. The TICAM2 physically bridged toll like receptor-4 (TLR4) with TICAM1 and the TLR4 partially regulated by the HIF during adenocarcinoma [54, 55].

All these results suggest that the cluster 6 represents the panel of either HIF or Hypoxia related lung cancer biomarkers.

Analysis of Cluster 7 / Panel 4

In the Cluster 7, there were seven lung biomarkers, mostly encoding for lung tissue specific extra cellular matrix proteins. The epigenetic analysis using Methycancer database (http://methycancer.genomics.org.cn) revealed that amongst the seven, KIAA1324, NET1, NTN3, RPL10 and TFPI2 were epigenetically regulated through DNA methylation. In the remaining two, SFTPA1 was epigenetically regulated [56–58]. However, the experimental evidence was lacking the epigenetic related data for CRISP3. However, the Gene card database analysis of CRISP3 showed that the CRISP3 orthlogous gene C-type lectin domain family 18 member A (CLEC18A) epigenetically regulated through DNA methylation (http://www.genecards.org). All these results show that the cluster 7 represented the panel of epigenetically regulated lung cancer specific extra cellular matrix biomarkers.