Modulation of epidermial growth factor receptor signaling in colon adenocarcinoma

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, monoclonal antibodies (mAbs) and EGFR tyrosine kinase inhibitors (TKIs) seemed to be the most promising. However they have demonstrated low utility in therapy, the former being effective at toxic doses, the latter resulting inefficient in colon cancer. This thesis work presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphtoquinone core as shikonin, an agent with great anti-tumor potential. In HT-29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 μM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer. In addition, surface plasmon resonance (SPR) investigation of the direct EGF/EGFR complex interaction using different experimental approaches is presented. A commercially available purified EGFR was immobilised by amine coupling chemistry on SPR sensor chip and its interaction to EGF resulted to have a KD = 368 ± 0.65 nM. SPR technology allows the study of biomolecular interactions in real-time and label-free with a high degree of sensitivity and specificity and thus represents an important tool for drug discovery studies. On the other hand EGF/EGFR complex interaction represents a challenging but important system that can lead to significant general knowledge about receptor-ligand interactions, and the design of new drugs intended to interfere with EGFR binding activity.

Phenotypic and molecular investigation of circulating tumor cells (CTCs) isolated from breast cancer patients at single cell resolution

Despite the paramount advances in cancer research, breast cancer (BC) still ranks one of the leading causes of cancer-related death worldwide. Thanks to the screening campaign started in developed countries, BC is often diagnosed at early stages (non-metastatic BC, nmBC), but disease relapse occurrence even after decades and at distant sites is not an uncommon phenomenon. Conversely, metastatic BC (mBC) is considered an incurable disease. The major perpetrators of tumor spread to secondary organs are circulating tumor cells (CTCs), a rare population of cells detectable in the peripheral blood of oncologic patients. In this study, CTCs from patients diagnosed with luminal nmBC and mBC (hormone receptor positive, Human Epidermal Growth Factor Receptor 2 (HER2) negative) were characterized at both phenotypic and molecular levels. To better understand the molecular mechanisms underlying their biology and their metastatic potential, next-generation sequencing (NGS) analyses were performed at single-cell resolution to assess copy number aberrations (CNAs), single nucleotide variants (SNVs) and gene expression profiling. The findings of this study arise hints in CTC detection, and pave the way to new application in CTC research.