The Role of c-Met as a Biomarker and Player in Innate and Acquired Resistance in Non-Small-Cell Lung Cancer: Two New Mutations Warrant Further Studies

In this video we discuss our retrospective study focused on the frequency of different c-Met aberrations (overexpression, amplification and mutations) in 153 primary, therapy-naïve resection samples and their paired metastases, from Biobank@UZA. Furthermore, we determined the correlation of c-Met expression with clinicopathological factors, Epidermal Growth Factor Receptor (EGFR)-status and TP53 mutations. Our results showed that c-Met expression levels in primary tumors were comparable to their respective metastases. Five different mutations were detected by deep sequencing: three (E168D, S203T, N375S) previously described and two never reported (I333T, G783E). I333T, a new mutation in the Sema(phorin) domain of c-Met, might influence the binding of antibodies targeting the HGF-binding domain, potentially causing innate resistance. E168D and S203T mutations showed a trend towards a correlation with high c-Met expression (p = 0.058). We found a significant correlation between c-MET expression, EGFR expression (p = 0.010) and EGFR mutations (p = 0.013), as well as a trend (p = 0.057) with regards to TP53 mutant activity. In conclusion this study demonstrated a strong correlation between EGFR mutations, TP53 and c-Met expression in therapy-naïve primary resection samples. Moreover, we found two new c-Met mutations that warrant further studies.

This article has been published on:
Molecules. 2019 Dec 4;24(24). pii: E4443. doi: 10.3390/molecules24244443.

Link: www.mdpi.com/1420-3049/24/24/4443

Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells

In this video we discuss how pancreatic stellate cells (PSCs) represent a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. Moreober we explain that c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. Effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids’ architecture was evaluated by confocal microscopy. PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC cells were more resistant to gemcitabine compared to homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. In conclusion, we developed spheroid models to evaluate PSC-PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.

This video has been published as supplemental file to the article:

Cancers (Basel). 2019 May 8;11(5). pii: E638. doi: 10.3390/cancers11050638
Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells
Firuzi O, Che PP, El Hassouni B, Buijs M, Coppola S, Löhr M, Funel N, Heuchel R, Carnevale I, Schmidt T, Mantini G, Avan A, Saso L, Peters GJ, Giovannetti E.
Link: www.ncbi.nlm.nih.gov/pubmed/31072019

Of note, the author of this paper, Professor Omidreza Firuzi (from the Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences , Shiraz, Iran), won the NWO Visitor’s Travel grant to work for one year in our lab.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this video is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

This video has been published as supplemental file to the article:

J Vis Exp. 2016 Dec 9;(118). doi: 10.3791/54714.
Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models
Sciarrillo R, Wojtuszkiewicz A, Kooi IE, Gómez VE, Boggi U, Jansen G, Kaspers GJ, Cloos J, Giovannetti E.

Link: www.ncbi.nlm.nih.gov/pubmed/28060337

Rociletinib: what went wrong?

In this video, the wide-ranging aspects of the evolution of EGFR-TKIs were described, with a special focus on rociletinib. The influence of different oncogenic mutations on EGFR activity was also discussed. Resistance to the first (erlotinib, gefitinib)- and second (afatinib)-generation EGFR-TKIs provided the rationale behind the development of the third-generation inhibitors (rociletinib, osimertinib). On the basis of these data, a comparison of their efficacy on the different mutated EGFRs and the respective resistance mechanisms is further reported. Moreover, the evolution and results of the clinical trials of rociletinib (TIGER trials) are compared with the trials on osimertinib, another third-generation EGFR-TKI that now has been granted US Food and Drug Administration approval. The reasons behind the arrest in the further development of rociletinib are put in the perspective of future drug development.

This video has been published as supplemental file to the article:

Onco Targets Ther. 2016 Oct 6;9:6065-6074. eCollection 2016.
New developments in the management of non-small-cell lung cancer, focus on rociletinib: what went wrong?
Van Der Steen N, Caparello C, Rolfo C, Pauwels P, Peters GJ, Giovannetti E
Link: www.ncbi.nlm.nih.gov/pubmed/27785053

Laser Micro-Dissection (LMD) & Genetic Analyses in New Preclinical Models to improve Pancreatic Cancer Treatment

Leica Laser Microdissection and Applications
From Eye to Insight

In this Webinar, after a technical presentation by Dr. Tamara Straube, Dr. Giovannetti discuss the advantages of using LMD techniques for precise, contamination-free isolation of specific cell types in various applications, such as using pancreatic cancer tissue sections from patients and orthotopic xenografts. Thus, this webinar provides an overview of the scientific and practical considerations for obtaining highly pure material for molecular analysis in the field of pharmacological studies to overcome key mechanisms of resistance in pancreatic cancer.

Aula Abierta: Liquid Biopsies

Talking about liquid biopsies, and how this type of analysis can be used for.
– Leticia G. Leon

Aula Abierta: Discussing real options to work as a researcher in Spain

Talking about the real possibilities to work in Spain as a researcher. The discussion presented different points of view. I explained why I had to move to another country to keep working in research.
– Leticia G. Leon

High-frequency ultrasound in orthotopic pancreatic cancer models

High-frequency ultrasound in Power Doppler Mode (Vevo 2100 imaging system, FujiFilm VisualSonics, thanks to the help of Dr. Dieter Fuchs) was carried out to evaluate tumor vascularization.
This high-resolution ultrasound instrument provided a non-invasive anatomical image of tumors or normal abdominal organs with an axial resolution of 40 μm, allowing multiple focal depths and improved feature detection. In particular, we observed hypovascular pancreatic tumors below 900 μm in diameter.

This video has been published as Supplemental file to the article:

Avan A, Caretti V, Funel N, Galvani E, Maftouh M, Honeywell RJ, Lagerweij T, Van Tellingen O, Campani D, Fuchs D, Verheul HM, Schuurhuis GJ, Boggi U, Peters GJ, Würdinger T, Giovannetti E.
Crizotinib inhibits metabolic inactivation of gemcitabine in c-Met-driven pancreatic carcinoma
Cancer Res. 2013 Nov 15;73(22):6745-56
Link: www.ncbi.nlm.nih.gov/pubmed/24085787

Laser-microdissection of pancreatic ductal adenocarcinoma tissues

Example of Laser-microdissection of pancreatic ductal adenocarcinoma tissues using the LMD7000 instrument (Leica Microsystems, Wetzlar, Germany, thanks to the help of Dr. Mauro Baron). The precision of the narrow focus of the laser beam allowed to pickup specimens with high degree of accuracy and extremely low risk of contamination.

This video has been published as supplemental file to the article:

Giovannetti E, Funel N, Peters GJ, Del Chiaro M, Erozenci LA, Vasile E, Leon LG, Pollina LE, Groen A, Falcone A, Danesi R, Campani D, Verheul HM, Boggi U.
MicroRNA-21 in pancreatic cancer: correlation with clinical outcome and pharmacologic aspects underlying its role in the modulation of gemcitabine activity
Cancer Res. 2010;70:4528-38
Link: www.ncbi.nlm.nih.gov/pubmed/20460539

Primary pancreatic cancer cell cultures

These cells and the methodology used for their isolation have been described in the article:

Funel N, Giovannetti E, Del Chiaro M, Mey V, Pollina LE, Nannizzi S, Boggi U, Ricciardi S, Del Tacca M, Bevilacqua G, Mosca F, Danesi R, Campani D.
Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma
Lab Invest. 2008 Jul;88(7):773-84.
Link: www.ncbi.nlm.nih.gov/pubmed/18490900