Lay Summary

Bile Tract Cancers (BTC) include cholangiocarcinoma (CCA) and gallbladder cancer (GBC) and their incidence is increasing worldwide [1, 2] . Lack of effective radical treatments highlights the need for a better understanding of BTC biology and mechanisms of response to treatment [1, 2]. Eighty-percent of BTC patients present at an advanced stage, when treatment options are limited to chemotherapy with cisplatin and gemcitabine (CG) [3, 4] . Only 11% of patients gain a long-term benefit from chemotherapy, while primary resistance is detected in 20% of patients (6). Most patients develop secondary resistance after an initial response or stabilization of the disease, which is responsible for a global median overall survival shorter than 12 months. Several mechanisms of chemo-resistance may act synergistically and drive cancer cells to escape biochemical inhibition or cell death caused by chemotherapy  [3, 4]. MicroRNAs (miRNA) are small non-coding-RNAs controlling mRNA expression [5]. The discovery of microRNAs (miRNAs) has opened up new avenues of research in biomedicine, in particular in cancer, and contributed to a large extent to the “Noncoding RNA revolution”. MiRNAs are key regulators of many cell processes often deregulated in cancer, including apoptosis. Several studies of the PI and others demonstrated that miRNAs are aberrantly expressed in BTC, promote biliary carcinogenesis and are involved in chemo-resistance [6-10]. Our group has recently demonstrated that miR-181a and b control target genes implicated in mitochondrial biogenesis, clearance, functionality and antioxidant response, uncovering a fundamental role of miR-181a and b in the control of autophagy/mitophagy, mitochondrial cell death and mitochondrial functionality [11, 12]. Notably mitochondria are emerging as important determinants of several aspects of cancer development and progression, including metabolic reprogramming, acquisition of metastatic capability, and response to chemotherapeutic drugs.

Altered expression of miR-181a has been discovered in several kinds of cancers, and it was significantly down-regulated in oral squamous cell carcinoma, while significantly up-regulated in thyroid cancer [13]. Moreover members of the miR-181 family have been investigated as prognostic biomarkers in Non-Small-Cell Lung Carcinoma (NSCLC), in Colorectal cancer (CRC) and up-regulation of miR-181 family members was recently shown to be associated with chemotherapy response in gastric cancer patients (which is treated with a combination of agents including cisplatin) [11]. Different studies have shown that aberrantly high miR-181a expression can regulate cell cycle, apoptosis, invasion, proliferation, and metastasis in several kinds of cancer [11] .

Given these observations, the important number of mechanisms with clinical relevance controlled by miR-181 family members and the association between the expression of these miRNA and the outcome of patients affected by distinct cancers, there is a strong demand to assess the prognostic significance of these microRNAs in multiple cancers. In particular there is a lack of systematic study showing the role of miR-181 family in the modulation of chemotherapy sensitivity and resistance in rare tumors such as BTC where resection of the primary tumour followed by chemotherapy still represent the first line clinical approach.

Executive Summary

1. Scientific Merit

A good clinical biomarker should have the following characteristics: preferentially produced in target tissue, data can be used to bridge nonclinical and clinical species, display early change for therapeutic intervention, detected through robust analytical assays and easily accessed through body fluids such as blood or urine. In this project we purpose to study the role of miR181 in sensitivity/resistance to cancer treatments and to develop and validate a new predictive miRNA biomarker in the treatment of rare tumours. It has been shown that microRNA expression is subject to change during the onset and progression of disease. This has led to the application of microRNAs as disease biomarkers. MicroRNAs are actively released from cells into the supernatant. This mechanism greatly facilitates the application of microRNAs as disease biomarkers. The presence of circulating microRNAs has been detected in various biofluids including serum/plasma, urine, and saliva.

Circulating microRNAs are released within vesicles as well as in association with RNA binding proteins and appear to be very stable in biofluids. Close to 50 % of the RNA contained in extracellular vesicles in serum and plasma are microRNAs. The remaining 50 % are other non-coding RNAs and to a small extent mRNAs.

Circulating microRNAs serve as non-invasive biomarkers for human diseases. MicroRNA biomarkers have utility for the following applications:

• toxicology: tissue health surrogate
• diagnosis: early diagnosis of asymptomatic diseases
• monitoring: detection of treatment response or adverse events

Recent studies and preliminary data strongly suggested a role of miR-181 family in chemotherapy sensitivity and resistance in Pancreatic-Biliary Cancers (see preliminary data). In patients with BTC the treatment goals is complete surgical resection (preserving organ function) and reduction of cytotoxic therapy by prospective risk stratification using biologic features. Although several prognostic factors have been defined (i.e. age, stage, histology, recurrent genetic anomalies), the identification of non-invasive biomarkers for disease follow-up and therapy monitoring is indeed still a clinical need.

Here we propose to identify and validate a microRNA 181-biomarker signature by a systematic approach. We will integrate a variety of high-throughput genomic techniques to prioritize candidate biomarkers specific for the treatment of bile tract cancer. We will validate the miRNA signature by using in vitro pre-clinical and clinical samples. We will definitively provide evidences of their clinical significance in human cancers. Using this systematic and innovative approach this project aims to develop and validate a new predictive biomarker ready to be introduced in clinical practice.

2. Feasibility

Preliminary data described in the following section, strongly support the feasibility of the project; several studies have demonstrated that miR-181 family members have a pivotal role in cancer, acting either as tumor suppressors or oncomirs and modulating a wide range of mRNA targets belonging to the main cancer-related pathways. Recent findings show the implication of miR-181 family members in different solid tumors and hematological malignancies. The miR-181 has been investigated as promising and effective candidate in the development of highly sensitive, noninvasive biomarkers for early NSCLC diagnosis (https://clincancerres.aacrjournals.org/content/23/17/5311). Furthermore, miR-181a has showed to be an useful diagnostic biomarker in differentiating follicular from papillary thyroid cancer.

The two specific aims we propose to achieve our objective build up on robust preliminary data generated in our laboratory that demonstrate the validity of our hypothesis. In addition, our preliminary work also demonstrates that the research group has all the necessary expertise and technology to timely achieve the proposed tasks.

The feasibility will be also assured by the available core facilities TIGEM is located in Pozzuoli and comprises 18 independent research groups with 5000 square meters converted in laboratories and research services. TIGEM’s research portfolio spans three strategic programs: Cell Biology of Genetic Diseases, Genomics Medicine, and Molecular Therapy. Research activity at TIGEM is supported by core facilities (http://www.tigem.it/core-facilities) that provide at TIGEM state-of-the-art technology as well as “house-keeping” assistance. Each core is supervised by a TIGEM investigator and is operated by specialized technical staff. Cores offer high-quality and rapid scientific and technical services that help to improve and speed up the work of TIGEM investigators. Each core is supervised by a TIGEM investigator and is composed of specialized technical staff. Cores facilities that will be critical to achieve the research goals of the proposed project include the High content Screening facility (HCS), the Next generation sequencing (NGS) Facility, Bioinformatics and Advanced microscopy and Imaging facility.

- The HCS facility makes use of two automated confocal microscopes (OPERA and Operetta systems from PerkinElmer), robotic liquid handling stations (Hamilton STARLet and PerkinElmer Zephir) to manipulate compound libraries, 96- and 384-well plate washers and dispensers, and dedicated cell culture room.

- The NGS facility (http://www.tigem.it/core-facilities/next-generation-sequencing-facility) performs in-house whole-exome sequencing (WES), RNA sequencing and preferential exome sequencing.

- Advanced Microscopy and Imaging (http://www.tigem.it/core-facilities/advanced-microscopy-and-imaging). Provides technical assistance, expertise, and qualitative and quantitative evaluation of microscopy data. The Core is composed of Light Microscopy (LM) and Electron Microscopy (EM) divisions.

Recently at TIGEM, an important role for TFEB and MiT-TFE genes in tumour growth [14] has been discovered showing a considerable expertise of the Institute in cell metabolism and autophagy and a cancer-related environment that will facilitate the proposed research. The feasibility will be also assured by core facilities that provide at TIGEM state-of-the-art technology as well as “house-keeping” assistance. Each core is supervised by a TIGEM investigator and is operated by specialized technical staff. Cores offer high-quality and rapid scientific and technical services that help to improve and speed up the work of TIGEM investigators.

3. Expertise

The proposed project will be developed with solid and lasting partnerships with International Cancer Clinical Centres (Hepatobiliary and Pancreatic oncology unit, Institute of Cancer Sciences, University of Glasgow, UK; Medical Oncology and Hematology Department together with Medical Diagnostic and Public Health, Modena Cancer Centre/University of Modena and Reggio Emilia, IT). The large network of scientific collaborations, developed by the applicant’s group with international teams of excellence, guarantees the best available expertise to carry out the programmed activities within the scheduled timeline. The PI is a molecular biologist with a strong track record in functional characterization of cancer-related non coding RNA. In particular He recently published several papers on the role of non coding RNA in drug-resistence of BTC.  Dr. Carotenuto joined TIGEM one year ago after 4 years’ experience at The Institute of Cancer Research, London, UK. In the group of Dr. Braconi and subsequently under the supervision of Paul Workman, he produced several publications in top-journals. As showed in the references list of this application [6, 8-10, 15], his research work led to the identification of novel biomarkers in PAN-BTC cancers. Dr. Carotenuto provided significant scientific and technical support for projects involving molecular (CRISPR, RNAi, library screening, PCR, WB, protein Assays, etc...) and cell biology techniques (from 2D cell, 3D cell, organoids culture, primary cell culture and co-culture to cell functional assays; from flow cytometry and FACS to microscopy and high content imaging techniques). Dr Carotenuto will be an important asset for this ambitious project considering his long experience in operating high-throughput screening genomic facilities combined with experience in cancer genomics and with the expertise and research facilities of the hosting institution. Recently Dr Carotenuto was awarded a Marie Skłodowska-Curie Research Fellow reintegration grant co-funded by Horizon 2020 and AIRC for a project regarding Bile Duct Cancer and drug resistance. The experience awarded by Dr Carotenuto between molecular and clinical cancer research contributes to guarantee the feasibility of the project. Co-applicant and collaborator are molecula and clinical scientist with proved experience in the field of both miRNA and clinical oncology of BTC.

The Co-applicant, dr. Indrieri A. identified the microRNA 181a and b as potential therapeutic target for mitochondrial mediated neurodegeneration. She showed that these miRNAs are involved in the global regulation of mitochondrial turnover in the central nervous system through the simultaneous and fine-tuning modulation of different mitochondria-related pathways. As first author, she has published 5 articles in international peer-reviewed journals, including high impact journals such as American Journal of Human Genetics and EMBO Molecular Medicine. ). Moreover, the applicant has also established several intra and extra mural collaborations, as well as successful accomplished different grant applications. She also supervised the work of other Post-doctoral fellows, PhD students, and technicians. In the 2017 she became Junior Principal Investigators at University Federico II of Naples continuing a strong collaboration with TIGEM and working on the development of strategy able to modulate miR-181 in vivo with the goal to produce and to provide a gene-independent RNA-based therapeutic product that could be then eventually translated into clinic.

The clinical research unit of Dr Salati, actively participates to national and international collaborative research programmes in biliary cancers, within consortia such as the European Network for the Study of Cholangiocarcinoma (ENSCCA) and the Cholangiocarcinoma Italian Study Group (GICO). Dr Salati is mainly focused on the study of hepato-pancreato-biliary cancers (HPB) and his research interests are moving in three main directions: 1) running national and international phase I, II, and III clinical trials investigating novel compounds against HPB malignancies, 2) studying tissue and circulating biomarkers as predictors of response/resistance and defining novel therapeutic targets in patients with HPB, 3) developing novel pre-clinical models for drug testing and study mechanisms of resistance. The ultimate goal of my research aimed to advance and personalize cancer care in HPB cancers following the paradigm of (forward and reverse) translational research. Dr Salati recently published several research manuscripts on the topic in internationally-recognized peer-reviewed Scientific Journals (Salati et al. European J Cancer 2019, Salati et al. Liver International 2019), and I contributed to the drafting of reviews and educational articles (Salati et al Semin Liver Dis 2019; Adeva et al Liv Intern 2019).

I have lately been awarded with the Fondazione Berlucchi Best Poster Prize at last Associazione Italiana Oncologia Medica (AIOM) Meeting (XXI Congresso Nazionale AIOM Rome 25-27 October 2019) for the work entitled “The Prognostic Nutritional Index (PNI) is an independent predictor of survival in advanced biliary cancers (ABC) receiving first-line chemotherapy (1L)".

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest