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- Material and Methods
- Supporting Information
Studies on the low-abundance transcriptome are of paramount importance for identifying the intimate mechanisms of tumor progression that can lead to novel therapies. The aim of the present study was to identify novel markers and targetable genes and pathways in advanced human gastric cancer through analyses of the low-abundance transcriptome. The procedure involved an initial subtractive hybridization step, followed by global gene expression analysis using microarrays. We observed profound differences, both at the single gene and gene ontology levels, between the low-abundance transcriptome and the whole transcriptome. Analysis of the low-abundance transcriptome led to the identification and validation by tissue microarrays of novel biomarkers, such as LAMA3 and TTN; moreover, we identified cancer type-specific intracellular pathways and targetable genes, such as IRS2, IL17, IFNγ, VEGF-C, WISP1, FZD5 and CTBP1 that were not detectable by whole transcriptome analyses. We also demonstrated that knocking down the expression of CTBP1 sensitized gastric cancer cells to mainstay chemotherapeutic drugs. We conclude that the analysis of the low-abundance transcriptome provides useful insights into the molecular basis and treatment of cancer.
Each year, almost one million people worldwide are diagnosed with gastric cancer, and more than 700,000 people die of this disease, representing ∼10% of cancer mortality globally. Despite advances in diagnostic imaging that have improved the early detection of gastric cancer, the advanced stages of the disease still has a poor prognosis. Recent advances in stratified medicine have improved therapeutic responses in advanced HER2-positive gastric cancer patients treated with trastuzumab; however, even in these cases, resistance develops rapidly, the benefit is transient, and most of these individuals eventually progress, mainly due to the selection of nonexpressing malignant cell clones.
A challenge for the application of functional genomics to cancer research is to identify the expression profile of the low-abundance transcriptome as a potential source of tumor-specific genes useful as biomarkers and targets. The screening of human cancerous tissues by whole gene expression analyses has identified mainly transcripts related to cell–ECM interactions and the cell cycle and other gene ontology groups that clearly represent highly abundant transcripts. Although RNA-Seq can provide an unbiased profile of a transcriptome, the broad dynamic range of gene expression levels still necessitates considerable over-sequencing to effectively sample the differentially expressed transcriptome.
Yet, a useful alternative is the use of a PCR-based suppressive subtractive hybridization that can equalize the abundance of cDNAs within the target samples enriching low-abundance and rare transcripts, followed by gene expression analysis.[7, 8] This approach has been used in very few studies to profile the low-abundance transcriptome in human hepatoma,[8, 9] breast and nasopharyngeal carcinomas and identify genes potentially associated with breast cancer progression. Here, we applied this procedure to identify low-abundance transcripts that were differentially expressed in human gastric adenocarcinomas compared with their paired adjacent noncancerous tissues. The vast majority of the differentially expressed low-abundance transcripts was not detected when the whole transcriptome was analyzed, leading to the identification of novel biomarkers, specific intracellular pathways and gene targets, that were aberrantly expressed in the cancer tissue. Further functional studies identified CTBP1 as a novel target for sensitization of gastric cancer cells to chemotherapeutic drugs.
- Top of page
- Material and Methods
- Supporting Information
The identification of the low-abundance transcriptome is of paramount importance in cancer research because subtle changes in the activity of few genes can lead to malignant transformation and tumor dissemination. In this work, we demonstrated that the analysis of the low-abundance transcriptome permitted the identification of novel genes that could serve as disease markers. Most importantly, this study led to the identification of specific intracellular signaling pathways, their aberrantly leading genes and potential novel druggable targets for improving the treatment of advanced gastric cancer.
Consistent with previous data[5, 22] whole transcriptome analysis identified differentially expressed genes with biological functions that were mainly associated with cell adhesion and cell-ECM interactions. Interestingly, less than 5% of the differentially expressed genes were shared between the low-abundance and the whole transcriptome. Further analysis by qRT-PCR validated 90 % of the genes differentially expressed in the low-abundance transcriptome of gastric cancer, demonstrating the robustness of this method. Moreover, studies at the protein level using TMA validated the overexpression of laminin α3 (LAMA3), one of the three subunits of Laminin-322 (Ln-332). There is no evidence in the literature of the involvement of Ln-332 in human gastric cancer; however, more recent studies have demonstrated that gastric cancer cell lines exhibit transcriptional silencing of LAMA3 due to promoter methylation. The LAMA3 staining was located in the cytoplasm of the malignant epithelial cells of gastric cancer samples, showing no evidence of expression silencing suggesting that this process is probably occurring following adaptation of cell lines to in vitro culture. The possibility that TTN may be a marker for premalignant lesions is proposed and warrants further investigation. In this regard, recent data suggested that TTN, the largest polypeptide encoded by the human genome might has an oncogenic role;[24-26] TTN is considered a protein kinase, and 63 non synonymous mutations were found in its coding regions in different cancer types of which half might be considered driver mutations.
The analysis of principal pathways in the low-abundance transcriptome highlighted intracellular signaling pathways that differed at a high extent from those obtained following whole transcriptome analyses. Among the intracellular signaling pathways selected for further validation by PCRArrays, only 2, 10, 6 and 5 genes exhibited more than 100-fold overexpression in the Wnt/hedgehog, PI3K/Akt, angiogenesis and B/T-cell activation intracellular pathways, respectively. These genes should be considered as leading the aberrant activities of the enriched pathways and then as preferable targets.
VEGF-C was at the top of the differentially expressed genes. VEGF-C has been associated with lymphatic spread and the invasion capacity of many types of cancer cells, including gastric cancer cells.[28, 29] IRS-2, a member of the PI3K/AKT signaling pathway was also highly expressed in gastric cancer samples; the differential abilities of IRS-1 and IRS-2 are intriguing. Although IRS-2 expression has been associated with lymph node metastasis in gastric cancer, loss of IRS-1 expression or function may facilitate tumor progression.[31, 32] Interestingly, IGF1, IGF2, insulin-like growth factor 2 receptor (IGF2R) and insulin (INS), all of which are components of the insulin/IGF signaling pathway, were also upregulated. The expression of IL-17 and IFNγ in association with the T/B cell activation pathway has been associated with the activation of the cytotoxic T and NK cell pathways in response to Helicobacter pylori infection. Interleukin-17 plays a potential role in the inflammatory response, and in agreement with the present data, its overexpression has been observed in gastric cancer samples. IFNγ is a classic pro-inflammatory cytokine secreted by NK cells and CD8 T-cells that has been associated with different tumor lineages.[37, 38] Among the large family of FZD receptors associated with the Wnt signaling pathway, we showed that FZD5 exhibited nearly 1,000-fold overexpression in gastric cancer samples. Its ligand, Wnt5a, also exhibited the largest differential expression among the different Wnt members between cancer and adjacent noncancerous samples. Wnt5a is involved in the activation of canonical and noncanonical Wnt cascades in epithelial cells located at the tumor-stromal interface during invasion and metastasis. Interestingly, pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor (TNFα) upregulate Wnt5a levels in gastric cancer, and Wnt5a overexpression correlated with a poor prognosis. In coincidence, the largest overexpression in the Wnt pathway was associated with WISP1. Recent studies have shown that WISP1 expression is coordinately regulated by Wnt5a and antagonists of the canonical Wnt pathway.
To functionally validate the data obtained with the low-abundance transcriptome analyses, we decided to perform additional studies with CTBP1. Knocking down the expression of CTBP1 inhibited the clonogenic and migration capacities of the cells, consistent with CTBP1's role as a mediator of hypoxia-induced tumor cell migration. Recent studies have shown that CTBP1 might act as a sensitizer to chemotherapeutic drugs, at least in breast cancer cells. Here, we show for the first time that knocking down the expression of CTBP1 sensitized AGS gastric cancer cells to three different chemotherapeutic drugs, 5-FU, cisplatin and epirubicin, decreasing their IC50s by 5-fold, 6-fold and 14-fold, respectively. The broad chemosensitizing effect of CTBP1 is supported by recent evidence that CTBP1 can promote drug resistance by increasing the expression of MDR1. Downregulation of CTBP1 expression in breast cancer cells renders malignant cells more sensitive to 5-FU and other genotoxic agents such as cisplatin and etoposide.21,
The development of molecular-targeted drugs for cancer treatment has demonstrated some success. However, even the most successful biological drugs are effective only in a minority of patients; occasionally, less that 10% of patients benefit from treatment. Here, we identified novel biomarkers and leading genes of signaling pathways that are aberrantly activated in gastric carcinomas that might provide the basis for increasingly specific targeted therapies.