Mirko Andreoli

βIII-Tubulin: biomarker of taxane resistance or drug target?

Introduction: βIII-Tubulin (TUBB3) is predominantly expressed in neurons of the central and peripheral nervous systems, while in normal non-neoplastic tissues it is barely detectable. By contrast, this cytoskeletal protein is abundant in a wide range of tumors. βIII-Tubulin is linked to dynamic instability of microtubules (MTs), weakening the effects of agents interfering with MT polymerization. Based on this principle, early studies introduced the classical theory linking βIII-tubulin with a mechanism of counteracting taxane activity and accordingly, prompted its investigation as a predictive biomarker of taxane resistance. Areas covered: We reviewed 59 translational studies, including cohorts from lung, ovarian, breast, gastric, colorectal and various miscellaneous cancers subject to different chemotherapy regimens. Expert opinion: βIII-Tubulin functions more as a prognostic factor than as a predictor of response to chemotherapy. We believe this view can be explained by βIII-tubulins association with prosurvival pathways in the early steps of the metastatic process. Its prognostic response increases if combined with additional biomarkers that regulate its expression, since βIII-tubulin can be expressed in conditions, such as estrogen exposure, unrelated to survival mechanisms and without any predictive activity. Additional avenues for therapeutic intervention could emerge if drugs are designed to directly target βIII-tubulin and its mechanism of regulation.

Novel Drugs Targeting Microtubules: the Role of Epothilones

Among the drugs targeting microtubule functions by interfering with tubulin subunits, epothilones represent a class of anticancer agents which recently entered clinical development. Although epothilones share mechanisms of action similar to taxanes, they have non-overlapping mechanisms of resistance; in particular, while overexpression of class III β-tubulin plays a major role in taxane resistance, epothilones display their highest efficacy in class III β-tubulin overexpressing malignancies. Three compounds belonging to this family (patupilone, ixabepilone and sagopilone), have been actively investigated in clinical trials, and some of them are at an advanced stage of development. This review provides a comprehensive analysis of the available literature on epothilones, focusing on their clinical development and potential as an additional weapon in the arsenal against tumors.

Class III β-tubulin in normal and cancer tissues

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.

Identification of the first inhibitor of the GBP1:PIM1 interaction. Implications for the development of a new class of anticancer agents against paclitaxel resistant cancer cells.

Class III β-tubulin plays a prominent role in the development of drug resistance to paclitaxel by allowing the incorporation of the GBP1 GTPase into microtubules. Once in the cytoskeleton, GBP1 binds to prosurvival kinases such as PIM1 and initiates a signaling pathway that induces resistance to paclitaxel. Therefore, the inhibition of the GBP1:PIM1 interaction could potentially revert resistance to paclitaxel. A panel of 44 4-azapodophyllotoxin derivatives was screened in the NCI-60 cell panel. The result is that 31 are active and the comparative analysis demonstrated specific activity in paclitaxel-resistant cells. Using surface plasmon resonance, we were able to prove that NSC756093 is a potent in vitro inhibitor of the GBP1:PIM1 interaction and that this property is maintained in vivo in ovarian cancer cells resistant to paclitaxel. Through bioinformatics, molecular modeling, and mutagenesis studies, we identified the putative NSC756093 binding site at the interface between the helical and the LG domain of GBP1. According to our results by binding to this site, the NSC756093 compound is able to stabilize a conformation of GBP1 not suitable for binding to PIM1.

Epstein-Barr Virus MicroRNA Expression Increases Aggressiveness of Solid Malignancies

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.

Integrated multidimensional analysis is required for accurate prognostic biomarkers in colorectal cancer.

CRC cancer is one of the deadliest diseases in Western countries. In order to develop prognostic biomarkers for CRC (colorectal cancer) aggressiveness, we analyzed retrospectively 267 CRC patients via a novel, multidimensional biomarker platform. Using nanofluidic technology for qPCR analysis and quantitative fluorescent immunohistochemistry for protein analysis, we assessed 33 microRNAs, 124 mRNAs and 9 protein antigens. Analysis was conducted in each single dimension (microRNA, gene or protein) using both the multivariate Cox model and Kaplan-Meier method. Thereafter, we simplified the censored survival data into binary response data (aggressive vs. non aggressive cancer). Subsequently, we integrated the data into a diagnostic score using sliced inverse regression for sufficient dimension reduction. Accuracy was assessed using area under the receiver operating characteristic curve (AUC). Single dimension analysis led to the discovery of individual factors that were significant predictors of outcome. These included seven specific microRNAs, four genes, and one protein. When these factors were quantified individually as predictors of aggressive disease, the highest demonstrable area under the curve (AUC) was 0.68. By contrast, when all results from single dimensions were combined into integrated biomarkers, AUCs were dramatically increased with values approaching and even exceeding 0.9. Single dimension analysis generates statistically significant predictors, but their predictive strengths are suboptimal for clinical utility. A novel, multidimensional integrated approach overcomes these deficiencies. Newly derived integrated biomarkers have the potential to meaningfully guide the selection of therapeutic strategies for individual patients while elucidating molecular mechanisms driving disease progression.

Adrenomedullin in Ovarian Cancer: Foe In Vitro and Friend In Vivo?

Stromal elements within a tumor interact with cancer cells to create a microenvironment that supports tumor growth and survival. Adrenomedullin (ADM) is an autocrine/paracrine factor produced by both stromal cells and cancer cells to create such a microenvironment. During differentiation of macrophages, ADM is produced in response to pro-inflammatory stimuli and hypoxia. In this study we investigated the role of ADM as a growth factor for ovarian cancer cells and as a modulator of macrophages. We also analyzed ADM expression levels in a retrospective clinical study using nanofluidic technology and assessment of ADM at the gene level in 220 ovarian cancer patients. To study the effects of ADM, ovarian cancer cell lines A2780, OVCAR-3, and HEY and their drug-resistant counterparts were used for proliferation assays, while monocytes from healthy donors were differentiated in vitro. ADM was a weak growth factor, as revealed by proliferation assays and cell cycle analysis. After culturing cancer cells under stressing conditions, such as serum starvation and/or hypoxia, ADM was found to be a survival factor in HEY but not in other cell lines. In macrophages, ADM showed activity on proliferation/differentiation, primarily in type 2 macrophages (M2). Unexpectedly, the clinical study revealed that high expression of ADM was linked to positive outcome and to cancer with low Ca125. In conclusion, although in vitro ADM was a potential factor in biological aggressiveness, this possibility was not confirmed in patients. Therefore, use of an ADM antagonist would be inappropriate in managing ovarian cancer patients.

GTP is an allosteric modulator of the interaction between the guanylate-binding protein 1 and the prosurvival kinase PIM1

GBP1 and PIM1 are known to interact with a molar ratio 1:1. GBP1:PIM1 binding initiates a signaling pathway that induces resistance to common chemotherapeutics such as paclitaxel. Since GBP1 is a large GTPase which undergoes conformational changes in a nucleotide-dependent manner, we investigated the effect of GTP/GDP binding on GBP1:PIM1 interaction by using computational and biological studies. It resulted that only GTP decreases the formation of the GBP1:PIM1 complex through an allosteric mechanism, putting the bases for the identification of new compounds potentially able to revert resistance to paclitaxel.

Herpes virus microRNA expression and significance in serous ovarian cancer.

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality.

Abstract 3127: MiR-BART9 is a prognostic biomarker associated with PD-1 expression in colon cancer patients

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 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 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.