Exosomal miRNA profiling to depict mechanisms of drug resistance in triple negative breast cancer - SUNRISE

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Project title: Exosomal miRNA profiling to depict mechanisms of drug resistance in triple negative breast cancer

Project acronym: SUNRISE

Competition: P1 – Development of the National R&D System – Postdoctoral research project (PD)

Code : PN-III-P1-1.1-PD-2021-0471

Financing contract nr. PD44/2022

Period : 01.04.2022 - 31.03.2024

Budget : 250.000 LEI

Coordinator : Research Center for Functional Genomics, Biomedicine and Translational Medicine from „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania

Project manager : CSIII Dr Jurj Maria-Ancuța

Mentor: Prof. Univ. Dr. Ciuleanu Tudor Eliade

Motivation of the proposed project

Nowadays, breast cancer is one of the most common malignancies, representing a leading cause of morbidity and mortality among women. An important subtype of breast cancer is triple negative breast cancer (TNBC). TNBC represents 15% to 20% of breast cancer cases, being associated with an increased mortality caused by the frequent development of resistance to chemotherapy and frequent relapses. In this context, an important role is played by the implication of tumor microenvironment and its communication with the malignant clone via exosomes. Exosomes are key elements in mediating intercellular communication, transporting biological molecules from donor to target cells. Exosomes can deliver to a target cell important biological molecules, such as nucleic acids (RNA, DNA, miRNA), lipids and proteins. Lately, microRNAs gained attention in the field of exosomes by offering a real mirror of the donor cell.
The aim of this project is the identification of new signaling pathways which can be targeted to counteract doxorubicin resistance in TNBC, as well as the determination of a potential biomarker to predict doxorubicin resistance in patients with TNBC.

Specific objectives of the project:

The project has the following objectives:

1. MiRNA analysis in exosomes isolated from resistant and sensitive TNBC cell lines and CAFs (cancer associated fibroblasts), focusing on the miRNAs altered pattern in relationship with drug resistance.
2. Determination of the effect that CAFs exhibit on sensitive TNBC cells biology, showing the modulation of doxorubicin resistance through the transfer of CAF-derived exosomes and soluble factors.

The project phases:

Phase 1: (period 31.12.2022) - Summary 2022

• MiRNA analysis in exosomes isolated from resistant and sensitive TNBC cell lines and CAFs, focusing on the miRNAs altered pattern in relationship with drug resistance.
• Determination of the effect that CAFs exhibit on sensitive TNBC cells biology, showing the modulation of Doxorubicin resistance through the transfer of CAF-derived exosomes and soluble factors.
• Evaluating the altered miRNAs in plasma-derived exosomes isolated from TNBC patients as a marker of therapy resistance prediction.
• Ethical committee approval.
• Project management.

The obtained results. Phase 1/ 2022

1. The isolation of exosomes from resistant and sensitive TNBC cell lines and CAFs through differential centrifugation. Physical and molecular characterization of the isolated exosomes
   The exosomes were isolated from cell culture media using ultracentrifugation at 109 000 x g for two hours at 4˚C. After centrifugation, exosomes were characterized through Nanosight (to determine the size and concentration of the exosomes), Transmission electron microscopy (to provide information about the morphology) and Western blot (to confirm the presence of the following exosome markers: CD9, CD63, CD81). The obtained results show that the extracellular vesicles which were isolated present specific features, such as the size of the particles is below 150 nm, "cup-shape" morphology as well as the presence of specific markers (CD9, CD63 and CD81). In this regard, we can assume that the obtained particles are exosomes and are suitable to be used for downstream evaluation.
2. The evaluation of altered miRNAs altered in TNBC and CAFs, using microarray.
   After characterization, RNA extraction from TNBC-derived exosomes was performed using Plasma/Serum Circulating and Exosomal RNA Purification Kit (Slurry Format) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific), while miRNA quality was assessed using an Agilent 2100 Bioanalyzer.
3. Validation of the most significant miRNAs in TNBC and CAFs - derived exosomes using qRT-PCR.
   Further, we evaluated the genes that were altered by the acquisition of doxorubicin resistance in MDA-MB-231 and Hs578T resulting in the preliminary selection of 31 genes. These genes was further evaluated in TCGA BRCA 2018 to determine which of them are associated with a change in the same direction in the disease-free survival (DFS) resulting in a total of 10 genes that we further evaluated in STRING. Among these 10 genes, we could see that only CXCL1, CXCL2, CXCL3, CXCL8, DUSP1, EGR1 and RRAD formed connections within the STRING network. Considering the literature, we observed that EGR1 is involved in multiple processes of interest in breast cancer including regulation of metabolism and migration of breast cancer cells. Thus, we assessed which microRNAs target EGR1 with a strong level of confidence and observed that 4 microRNAs met this criterion: miR-124-3p, miR-181a-5p, miR-183-5p, and miR-191- 5 p. These microRNAs will be analyzed in exosomes isolated from doxorubicin sensitive and resistant TNBC lines, as well as from CAFs.
4. Ethical committee approval.
   The appropriate documentation was developed (working protocol, informed consent template, standard application for CE approval), according to the university's standard operating procedures, as well as international legislation.
5. Project management
   The main aspects monitored within the management plan are: monitoring the performance of activities according to the project proposal, identifying gaps with respect to the terms of completion and finding solutions to reduce them, monitoring compliance with the budget allocated to this stage, effective communication both on scientific topics with the mentor and on administrative matters with the financial officer.
6. Web site
   Presentation material has been selected for the project web page, which will be updated with information at each phase.

SUMMARY OF THE PERFORMED ACTIVITIES

According to the main objective "MiRNA analysis in exosomes isolated from resistant and sensitive TNBC cell lines and CAFs, focusing on the miRNAs altered pattern in relationship with drug resistance", in the first phase of the project, an important emphasis was put on optimizing the isolation protocol of extracellular vesicles, especially exosomes, secreted by TNBC cell lines resistant and sensitive to doxorubicin, as well as CAFs. Subsequently, extracellular vesicles isolated from breast cancer cell lines were physically and molecularly characterized.

Until now, it has been shown that exosomes are remarkable entities involved in modulation of various biological processes, of which drug resistance is of great interest. The activation of drug resistance mechanisms is altered by the tumor microenvironment through several players, including extracellular vesicles, soluble factors, CAFs, epithelial cells, immune cells, etc. CAFs influence the tumor microenvironment and the malignant clone, making it more suitable for the development of the malignancy. Thus, the further evaluation of these processes can offer a better view on the acquisition of therapy resistance, leading both to the development of new predictors of resistance, but also of potential therapies that could reverse doxorubicin resistance.

Phase 2 (period 31.12.2023) - Summary 2023

• Evaluation of altered miRNAs altered in TNBC and CAFs, using microarray.
• Determination of the effect that CAFs exhibit on sensitive TNBC cells biology, showing the modulation of Doxorubicin resistance through the transfer of CAF - derived exosomes and soluble factors.
• Evaluating the altered miRNAs in plasma-derived exosomes isolated from TNBC patients as a marker of therapy resistance
• Ethical committee approval.
• Project management and data dissemination.

The obtained data in the project. Phase 2/ 2023

The obtained data in the project. Phase 2/ 2023

(1) Transcript expression levels were evaluated using the microarray platform. The Human miRNA Microarray Kit (version 21.0; format, 8x60LK slides) was employed for microarray analysis. Subsequently, samples were scanned with the Agilent Microarray G2565BA scanner (Agilent Technologies, Inc.). The data analysis encompassed the use of Feature Extraction software (version 10.7, Agilent Technologies, Inc.) and GeneSpring GX software version 12.6.1 (Agilent Technologies, Inc.).

Genes exhibiting altered expression levels, including both up- and down-regulated, were identified for each experimental condition through the GeneSpring program. The findings were as follows:
• For the Hs578T cell line in co-culture with fibroblasts: 183 genes were down-regulated, while 486 genes were up-regulated.
• In the case of the Hs578T cell line resistant to doxorubicin therapy in co-culture with fibroblasts: 417 genes showed down-regulation, and 1405 genes demonstrated up-regulation.
• Regarding the MDA-MB-231 cell line in co-culture with fibroblasts: 1778 genes were up-regulated, and 10588 genes exhibited overexpression.
• For the MDA-MB-231 cell line resistant to doxorubicin therapy in co-culture with fibroblasts: 1927 genes were down-regulated, while 13853 genes were up-regulated.

Following the bioinformatic analysis, attention was focused on six genes with altered expression levels: ABCB1, IL6, TGF-β1, CXCL11, ZEB1, and MALAT1.

(2) The subsequent phase of this study involves assessing the expression levels of genes implicated in therapy resistance mechanisms. Through bioinformatics analyses, genes displaying altered expression patterns were pinpointed. ABCB1, CXCL11, IL6, TGF-β1, ZEB1, and MALAT1 were specifically selected from this pool and subjected to analysis using the RT-qPCR technique in triple-negative breast cancer cell lines. These lines included those sensitive and resistant to doxorubicin therapy, cultivated in co-culture with fibroblasts. Post RT-qPCR analysis, the selected genes were validated, revealing their significant involvement, particularly in mechanisms governing resistance to therapy. Notably, individuals diagnosed with breast cancer exhibited diminished survival rates associated with the identified six genes.

Furthermore, an examination of the expression levels in patient samples, comparing normal to primary tumor tissues, disclosed a noteworthy decrease in ABCB1, IL6, and ZEB1 genes. In contrast, CXCL11, TGF-β1, and MALAT1 genes displayed an elevated expression profile. Consequently, these genes emerge as potential specific biomarkers for diagnostic and prognostic purposes, shedding light on their role in breast cancer.

(3) The exploration of microRNAs (miRNAs) influencing the modulation of resistance mechanisms to therapy through the regulation of previously mentioned genes constitutes a pivotal aspect of this investigation. Four miRNAs—miR-19b, miR-21, miR-125a, and miR-155—were deliberately chosen for their critical roles in modulating therapy resistance. Importantly, these selected miRNAs are direct targets of the aforementioned genes. An elevation in the expression levels of the investigated miRs was noted, indicating an activation of therapy resistance mechanisms. Furthermore, the presence of the tumor microenvironment, encompassing fibroblasts and exosomes, emerged as a crucial factor bolstering and triggering resistance mechanisms through diverse transcripts and cytokines.

Utilizing the TCGA database, a correlation was established between the overexpression of miR-21 and miR-155 and decreased survival rates in individuals diagnosed with breast cancer. Conversely, the underexpression of miR-125a was associated with a significantly diminished survival rate. Consequently, these miRNAs hold promise as specific biomarkers for both diagnostic and prognostic purposes, offering valuable insights into their potential role in breast cancer outcomes.

Phase 3 (period 31.03.2024) - Summary 2024

• Evaluating the altered miRNAs in plasma-derived exosomes isolated from TNBC patients as a marker of therapy resistance.

The obtained data in the project. Phase 3/ 2024

(1). We collected and processed blood samples from patients diagnosed with breast cancer. Upon obtaining informed consent, blood samples were collected in EDTA-containing tubes and processed within 30-60 minutes. The plasma was then extracted and stored at -80 degrees Celsius until further investigations were conducted.

(2). We isolated and characterized exosomes from the plasma of breast cancer patients. Exosomes were isolated using the ultracentrifugation method and characterized using NanoSight and Transmission electron microscopy. These methods revealed the size of the exosomes (<200nm) and their characteristic cup-shaped morphology.

(3). We analyzed miRNAs in exosomes isolated from plasma using RT-qPCR. After characterizing the exosomes, we evaluated miR-19b, miR-21, and miR-125a. We observed increased expression levels of these miRNAs in breast cancer cell lines, indicating activation of therapy resistance mechanisms and various biological processes such as tumor growth, invasion, and metastasis. Additionally, the tumor microenvironment, including fibroblasts and exosomes, supports and activates these therapy resistance mechanisms. Thus, these miRNAs may serve as specific biomarkers for diagnosis and prognosis.

The impact of the data

The proposed project is based on highlighting changes in communication between TNBC cells and CAFs in the context of doxorubicin resistance. By identifying the signaling pathways involved in modulating this process, we can determine key molecules to help predict the activation of therapy resistance mechanisms.

Dissemination

The manuscript has been published in the journal Translational Oncology https://doi.org/10.1016/j.tranon.2024.101951 

Contact

CSIII Dr Jurj Maria-Ancuta
Research Center for Functional Genomics, Biomedicine and Translational Medicine from „Iuliu Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania
E-mail: