Papel de Gasdermina B en terapia antitumoral en cáncer de mama HER2+

Sánchez Álvarez, Rocío (2019). Papel de Gasdermina B en terapia antitumoral en cáncer de mama HER2+. Proyecto Fin de Carrera / Trabajo Fin de Grado, E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM), Madrid.

Description

Title: Papel de Gasdermina B en terapia antitumoral en cáncer de mama HER2+
Author/s:
  • Sánchez Álvarez, Rocío
Contributor/s:
  • Sarrió López, José David
  • Echavarri Erasun, Carlos
Item Type: Final Project
Degree: Grado en Biotecnología
Date: July 2019
Subjects:
Faculty: E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM)
Department: Biotecnología - Biología Vegetal
Creative Commons Licenses: Recognition - No derivative works - Non commercial

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Abstract

Around 20% of invasive breast cancers show over-expression and/or amplification of HER2/neu oncogene and are therefore called “HER2 positive” (HER2+). HER2 over-expression/activation leads to tumor formation, enhanced proliferation and cancer survival, thus producing an aggressive behavior. These tumors are standardly treated with anti-HER2 drugs that block HER2 pro-tumor activities, such as the trastuzumab antibody and the tyrosine kinase inhibitor lapatinib, but unfortunately, many tumors exhibit mechanisms of resistance to these drugs. The main cause of HER2 over-activation in cancer is the amplification of the chromosomal region 17q12-21 containing HER2 oncogene as well as 10 to 27 additional genes. Among these frequently coamplified genes, our laboratory identified Gasdermin-B (GSDMB) over-expression (occurring in >60% of HER2 cancers) as a marker of poor prognosis, and reduced response to therapy in these tumors. Moreover, we demonstrated that GSDMB induces multiple pro-tumor functions, including the promotion of migration, invasion, metastasis as well as resistance to the anti-HER2 agents lapatinib and trastuzumab. GSDMB belongs to the Gasdermin (GSDMs) family of proteins, which are functionally involved in triggering cell death. These cytoplasmic proteins (6 members in humans) share a similar structure, consisting in an N-terminal domain with pro-cell death activity, a C-terminal auto-inhibitory domain, and a linker region. It has been demonstrated that GSDMD and GSDME can trigger pyroptosis, a type of lytic and pro-inflammatory cell death characterized by pore formation and cell membrane rupture. Moreover, recent evidences suggest that all GSDM members may also share this function. The activation of GSDMs depends on the release of the pore-forming N-terminal domain after the cleavage of the inter-domain linker region by specific caspases. Interestingly, after apoptotic induction by chemotherapeutic agents, active caspase-3 can cleave diverse GSDMs, producing either activation (GSDME) or inhibition (GSDMD) of their pyroptotic function. GSDMB can also be cleaved by caspase-3 in vitro, although the biological function of this processing is still unknown. In fact, the implication of GSDMB in pyroptosis is very controversial and data in the literature are inconsistent. Given the seemingly contradictory effects of GSDMB, on one hand promoting cell survival in cancer cells after anti-HER2 treatment, an on the other hand possibly having intrinsic pyroptotic function, our lab is interested in deciphering the molecular mechanisms mediated by GSDMB in tumors. Accordingly, to shed light into the mechanisms of GSDMB-mediated drug resistance, in this work we proposed two main objectives: a) Assess if GSDMB could promote survival to anti-HER2 drugs (lapatinib) by regulating the expression or activation of other GSDMs with known pyroptotic function (GSDMD and GSDME). b) Evaluate the utility of murine HER2/GSDMB+ breast cancer primary cultures, generated from transgenic mice, as pre-clinical models to mimic the multiple functions of GSDMB in human HER2 tumors. To achieve the first aim, we utilized two complementary models of human HER2 breast cancer cell lines in which GSDMB had been modified either by shRNA-silencing (HCC1954 cells) or by exogenous over-expression (SKBR3). We treated these models with lapatinib or DMSO (control) and analyzed the expression and cleavage of GSDMB, D, and E during apoptosis (and caspase-3) induction. Our results confirmed that lapatinib induced apoptosis and caspase-3 activation, which in turn cleaved GSDMB, D and E. However, we did not observe a consistent effect of GSDMB on the levels or the activation of the other GSDMs. These results, together with previous observation from our lab, suggest that GSDMB may promote survival to anti-HER2 agents independently of the pro-cell death function of other GSDMs. Regarding the second aim, it should be noticed that mice do not have a GSDMB orthologue gene. Thus, to create an in vivo animal model that could mimic the effects of GSDMB on HER2 human cancer patients, the lab had previously generated a novel transgenic mouse strain that expresses human GSDMB together with HER2 in spontaneous breast tumors. From these mice, a HER2/GSDMB+ breast cancer primary culture (GSCFU9di) was obtained. From the parental cells, the lab generated through stable shRNA-silencing diverse cellular models with different levels of GSDMB. In this work, we tested if GSDMB in these mouse cell models reproduced the same functions as those observed in human HER2 tumors, in particular its effect on survival after anti-HER2 treatment. Consistent with this hypothesis, our results showed that stable silencing of GSDMB did not affect the morphology or proliferation of GSCFU9di cells. In addition, the nucleus-cytoplasmic localization of GSDMB in these models replicated the localization found in GSDMB-overexpressing human tumors. All these characteristics indicate that GSDMB exhibits similar biological properties in human and mouse cancer cells. Unfortunately, contrary to human cancers, we did not observe a significant effect of GSDMB on the sensitivity of GSCFU9di cells to lapatinib. Hence, this mouse cell model does not reproduce the molecular mechanisms required for GSDMB-mediated drug resistance in HER2 tumors. In conclusion, although further studies are needed, our results suggest that in human cancer cells, GSDMB may promote survival to anti-HER2 agents independently of other GSDMs. Moreover, our mouse HER2 cell lines seem not to replicate the drug survival effect observed in human cells. To overcome this possible limitation other in vivo models are currently under evaluation such as patient derived xenografts.

More information

Item ID: 57135
DC Identifier: http://oa.upm.es/57135/
OAI Identifier: oai:oa.upm.es:57135
Deposited by: Biblioteca ETSI Agrónomos
Deposited on: 30 Oct 2019 15:11
Last Modified: 30 Oct 2019 15:11
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