Mechanisms of resistance toT-DM1 in HER2-positive breast cancer

Abstract

El cáncer de mama HER2-positivo representa alrededor del 15-30% del total de pacientes con cáncer de mama. Este subtipo tiene un mal pronóstico, seguido del subtipo Triple-negativo (TNBC), que lo tiene aún peor. Actualmente, varios medicamentos están aprobados para las pacientes HER2-positivo, como Trastuzumab, Lapatinib y T-DM1. Sin embargo, a menudo éstas adquieren resistencia a estas terapias en un período de 1 a 2 años. El objetivo principal de este estudio es definir los mecanismos de resistencia a T-DM1. Durante mi tesis, generé líneas celulares resistentes a T-DM1 y exploré diferentes posibilidades in vitro que pudieran explicar cómo estas células escapan de la terapia contra HER2. Obtuve células resistentes a T-DM1 usando una línea celular derivada de un tumor primario de una paciente (PDX, Patient-derived xenograft) tratándolas con dosis crecientes de T-DM1. Comprobé los niveles de HER2 por mRNA y proteína, número de copias, efectores de señalización, analicé la región citotóxica de T-DM1 y su actividad lisosómica diferencial comparando la línea parental y las células resistentes. Esta tesis describe punto por punto todos los posibles mecanismos de resistencia que investigamos y cuales resultaron ser los responsables de la resistencia a T-DM1.

HER2-positive breast cancer represents around 15-30% of the breast cancer patients. This breast cancer subtype has poor prognosis, followed by the triple negative subtype, which has the worse. Several drugs are currently approved for HER2-positive breast cancer, such as Trastuzumab, Lapatinib and T-DM1. However, often these patients acquire resistance to these therapies in a period of 1 - 2 years. The main goal of this study is defining mechanisms of resistance to T-DM1. First, I have generated T-DM1 resistant cell lines, and I explored in vitro different possibilities to explain how these cells escape from HER2-target therapy. I obtained T-DM1 resistant cells using a PDX-derived cell line (PDX118) through continuous treatment of increasing doses of T-DM1. These cells were tested of resistance to T-DM1 in the presence of this HER2 target drugs. I have tested whether the levels of HER2 by mRNA and protein, copy number, downstream signaling effectors, cytotoxic part of T-DM1 or differential lysosome activity might justify the differences between parental and resistant cells. No differences in copy number, neither at transcript or total protein levels of HER2 was observed in parental versus resistant cells. However, in two of three T-DM1 resistant cultures, the protein levels at the surface of the resistant cells were significantly lower, although the downstream signaling activity remained similar. A HER2 rescue experiment resulted in a partial recovery of the sensibility. HER2 protein levels at the surface of the tumor cells is the main mechanism of resistance to T-DM1 therapy, and also, abnormal function of lysosomes maybe a reason to escape this treatment.