Microtubules are polymeric structures composed of tubulin subunits. Each subunit consists of a heterodimer of α- and β-tubulin. At least seven β-tubulin isotypes, or classes, have been identified in human cells, and constitutive isotype expression appears to be tissue specific. Class III β-tubulin (βIII-tubulin) expression is normally confined to testes and tissues derived from neural cristae. However, its expression can be induced in other tissues, both normal and neoplastic, subjected to a toxic microenvironment characterized by hypoxia and poor nutrient supply. In this review, we will summarize the mechanisms underlying βIII-tubulin constitutive and induced expression. We will also illustrate its capacity to serve as a biomarker of neural commitment in normal tissues and as a pure prognostic biomarker in cancer patients.

The Cancer Genome Atlas (TCGA) microRNA (miRNA) initiative has revealed a pivotal role for miRNAs in cancer. Utilizing the TCGA raw data, we performed the first mapping of viral miRNA sequences within cancer and adjacent normal tissues. Results were integrated with TCGA RNA-seq to link the expression of viral miRNAs to the phenotype. Using clinical data and viral miRNA mapping results we also performed outcome analysis. Three lines of evidence lend credence to an active role of viral miRNAs in solid malignancies. First, expression of viral miRNA is consistently higher in cancerous compared to adjacent noncancerous tissues. Second, viral miRNA expression is associated with significantly worse clinical outcome among patients with early stage malignancy. These patients are also featured by increased expression of PD1/PD-L1, a pathway implicated in tumors escaping immune destruction. Finally, a particular cluster of EBV-miRNA (miR-BART2, miR-BART4, miR-BART5, miR-BART18, and miR-BART22) is associated with expression of cytokines known to inhibit host response to cancer. Quantification of specific viral miRNAs may help identify patients who are at risk of poor outcome. These patients may be candidates for novel therapeutic strategies incorporating antiviral agents and/or inhibitors of the PD-1/PD-L1 pathway.

Background: Epstein-Barr Virus (EBV) is widely expressed in the population in a latent stage. During cancer progression, antiviral immunity is compromised by the same mechanisms leading to suppression of anticancer immunity. This study was aimed at developing the expression of miR-BART as biomarkers to select patients at risk of fast progression for a defect of the antiviral/anticancer immunity. Methods: We first analyzed the expression of EBV-miRNAs in a panel of 438 colon cancer (CC) patients of the TCGA dataset. Analysis was conducted using level 1 miRNA-seq and level 3 RNA-seq available on the same patients. In order to confirm the results, we tested the expression of miR-BART9 in an additional set of 271 CC patients using spectral imaging and quantitative fluorescent immunohistochemistry (PD-1 and PD-L1 antigens at the protein level and miR-BART9 using a specific designed ISH probe). In order to increase the clinical applicability of our findings, we also developed a qPCR test to analyze the expression of miR-BART9 in serum of colon cancer patients. Results: In the analysis of 438 CC patients of the TCGA dataset, expression of miR-BART9 was found in 78 patients (18%). Levels of miR-BART9 were prognostic of poor outcome in a multivariate Cox model including age and stage (HR = 2.8, CI 1.6-4.7, p<0.0001). In patients positive for miR-BART9 we also noticed a significant higher expression of PD-1, CD3 but not PD-L1 at the gene level. These results suggest that the expression of miR-BART9 is linked to a T cell infiltrate in the cancer tissue capable to induce a potent suppression of anticancer and antiviral immunity. In order to confirm this hypothesis, we performed spectral imaging in an additional set of 271 CC patients. Expression of miR-BART9 was found in 32 (12%) patients with a prevalent stromal pattern. Also in this case expression of miR-BART9 was associated with poor outcome in Cox-multivariate analysis including age and stage (HR = 2.5, CI 1.6-3.4, p<0.001). Expression of miR-BART9 was again significantly associated with a PD-1 positive T-cell infiltrate. Based on these results, we have designed a qPCR test to analyze the expression of miR-BART9 in serum using an EBV-infected B lymphoma cell line (Raji) as positive control. The assay was employed for monitoring miR-BART9 expression in serum from CC patients (n = 89) and healthy controls (n = 95). Human miR-486 and miR-17 were used as positive controls.MiR-BART9 expression was found in 33 (37%) of CC patients and only in 5 (5%) of the 95 healthy controls. Conclusions: Our results reveal that expression of miR-BART9 is associated with aggressive CC. Patients featuring reactivation of latent EBV-infection have also expression of PD-1+ T cells, which will impact both anticancer and antiviral immunity. The expression of miR-BART9 can be detected also in serum and may serve to identify the right timing to treat CC patients with PD-1/PD-L1 inhibitors in order to restore an effective anticancer and antiviral immunity. Citation Format: Marisa Mariani, Deep Pandya, Mirko Andreoli, Shiquan He, Manuela Spennato, Cristiano Ferlini. MiR-BART9 is a prognostic biomarker associated with PD-1 expression in colon cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3127.