Research Article: Identification of a serum-induced transcriptional signature associated with metastatic cervical cancer

Date Published: August 30, 2017

Publisher: Public Library of Science

Author(s): Anna Palatnik, Shuyun Ye, Christina Kendziorski, Marissa Iden, Jessica S. Zigman, Martin J. Hessner, Janet S. Rader, Klaus Roemer.

http://doi.org/10.1371/journal.pone.0181242

Abstract

Tumor cells that escape local tissue control can convert inflammatory cells from tumor suppressors to tumor promoters. Moreover, soluble immune-modulating factors secreted from the tumor environment can be difficult to identify in patient serum due to their low abundance. We used an alternative strategy to infer a metastatic signature induced by sera of cervical cancer patients.

Sera from patients with local and metastatic cervical cancer were used to induce a disease-specific transcriptional signature in cultured, healthy peripheral blood mononuclear cells (PBMCs). An empirical Bayesian method, EBarrays, was used to identify differentially expressed (DE) genes with a target false discovery rate of <5%. Ingenuity Pathway Analysis (IPA) software was used to detect the top molecular and cellular functions associated with the DE genes. IPA and in silco analysis was used to pinpoint candidate upstream regulators, including cancer-related microRNAs (miRNAs). We identified enriched pathways in the metastatic cervical group related to immune surveillance functions, such as downregulation of engulfment, accumulation, and phagocytosis of hematopoietic cells. The predicted top upstream genes were IL-10 and immunoglobulins. In silco analysis identified miRNAs predicted to drive the transcriptional signature. Two of the 4 miRNAs (miR-23a-3p and miR-944) were validated in a cohort of women with local and metastatic cervical cancer. This study supports the use of a cell-based assay that uses PBMC “reporters” to predict biologically relevant factors in patient serum. Further, disease-specific transcriptional signatures induced by patient sera have the potential to differentiate patients with local versus metastatic disease.

Partial Text

The most significant prognostic factor for poor survival rates in patients with invasive cervical cancer is metastasis [1]. Unfortunately, metastasis can be difficult to identify and treat, and it does not directly relate to tumor size. Some patients with stage I disease, and even some with small and seemingly localized tumors, have metastases identified in lymph nodes or distant organs [1]. Consequently, identifying biologic pathways and biomarkers through novel techniques could improve detection and treatment for women with cervical cancer.

We used sera from women with localized (n = 5) or metastatic (n = 5) cervical SCC to drive transcription in healthy PBMC “reporters” and read the responses using genome-wide expression profiling. The transcriptional responses induced by the 11 (10 cancer and 1 healthy control) experimental samples were normalized using Robust Multi-Array Analysis (RMA) to obtain summary scores of expression for each profile set on each array [13]. We then used EBarrays to identify genes whose expression differed when comparing PBMCs stimulated by serum from women with localized versus metastatic disease. A total of 557 differentially expressed probe sets that mapped to 382 genes (of which 323 were unique) exhibited DE with a FDR of <5%. In the unique gene set, 184 were upregulated and 139 were downregulated in serum from women with metastatic disease. The most significant upregulated and downregulated genes from the patients with metastatic cervical cancer are shown in Table 3. The current study adopted a novel cell-based assay that used reporter PBMC cultures to characterize transcriptional signatures induced by serum from women with cervical cancer. Collectively, the results suggest that factors present in serum from women who have metastatic cancer rather than local disease associate with downregulation of immune-surveillance functions such as engulfment, accumulation, and phagocytosis of hematopoietic cells. Enrichment and pathway analyses suggest that the top candidates driving the serum effects include IL-10, immunoglobulin, and cellular responses to LTA and S. aureus. Although many elements in serum could account for these results, we focused our validation analysis on potential upstream miRNA candidates. In silico analysis revealed candidate miRNAs which could be responsible for driving the DE gene profiling results. Four of the top miRNA candidates were further probed for validation via qPCR on serum samples from a larger cohort of patients. We found that 2 out of the 4 were differentially expressed in serum from patients with metastatic versus local disease.   Source: http://doi.org/10.1371/journal.pone.0181242

 

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