Leandro S. D’Abronzo

Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis.

Abstract 1303: In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer

Background: Prostate cancers (PCa) rely on androgenic ligands and the androgen receptor (AR) for their growth and survival, making AR inhibition a predominant therapeutic strategy for these tumors. Some prostate tumors however fail this therapy due to ‘bypass’ mechanisms that emerge as a result of prolonged AR targeting. This in vivo study attempted to assess the expression and activation of the epidermal growth factor receptor (EGFR) family (whose role is well-documented in PCa) in response to androgen deprivation therapy (ADT). Methods: Nude mice were implanted (s.c.) with CWR22 tumors (human-patient-derived, androgen-dependent ‘AD’) and its castration-resistant (‘CR’) subline CWR22-Rv1 (relapsed CWR22). Androgen deprivation (i.e. AR inhibition) was achieved by surgical or ‘sham’ castration of mice. Tumors were analyzed (immunohistochemistry/immunoblot) for EGFR/ErbB2/ErbB3/AR proteins and proliferative/apoptotic markers. Results: Castration caused significant tumor regression in AD but not CR tumors. in vitro viability assays demonstrated that castration (mimicked by using charcoal-stripped serum, ‘css’) did not slow down CR cells to the same degree as it did AD cells. At baseline, intratumoral EGFR protein was unchanged in R22 tumors, ErbB2 levels decreased and ErbB3 protein increased in Rv1 tumors. Castration increased ErbB3 but not EGFR or ErbB2 proteins in CWR22 tumors. Phosphorylated forms of these receptors were generally difficult to detect but there was more phosphorylated ErbB3 protein in Rv1 tumors. Downstream of the EGFR family, there was less phosphorylated Erk but not Akt protein in CWR22-Rv1 tumors. Castration decreased Erk protein in AD tumors but increased it in CR tumors. Immunohistochemical quantification revealed that cytoplasmic EGFR and ErbB3 proteins were elevated in CR tumors but reduced in AD tumors. Castration greatly decreased Ki-67 staining in AD but not in CR tumors while the number of TUNEL-positive nuclei and intensity of PARP staining decreased in castrated CR but not in AD tumors. ErbB3 and AR proteins were significantly correlated with DNA damage and proliferation in CWR22 tumors but only nuclear AR levels and proliferation were significantly correlated in CR tumors. Conclusions: We conclude that androgen deprivation therapy may alter EGFR and ErbB3 protein levels and localization in androgen-dependent and castration-resistant tumors. The EGFR family is typically activated at the cell surface hence their presence and activity there, in response to castration, may initiate signalling pathways encouraging tumor cell proliferation and survival. Citation Format: Maitreyee K. Jathal, Thomas M. Steele, Salma Siddiqui, Benjamin A. Mooso, Leandro S. D’Abronzo, Christiana M. Drake, Paramita M. Ghosh. In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1303.

Evaluation of Protein Levels of the Receptor Tyrosine Kinase ErbB3 in Serum

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTK) consists of four members: EGFR1/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and HER4/ErbB4. Signaling through these receptors regulates many key cellular activities, such as cell division, migration, adhesion, differentiation, and apoptosis. The ErbB family has been shown to be overexpressed in different types of cancers and is a target of several inhibitors already in clinical trials. ErbB3 lacks a functional tyrosine kinase domain and therefore has not been as extensively studied as the other members of this family, but its importance in activating downstream pathways, such as the PI3K/Akt pathway, makes this RTK a worthy investigation target, especially in urothelial carcinoma where the PI3K/Akt pathway is vital for progression. In recent times, ErbB3 overexpression has been linked to drug resistance and progression of various diseases, especially cancer. ErbB3 levels in the serum were shown in many cases to be reflective of its role in disease progression, and therefore detection of serum ErbB3 levels during treatment may be of importance.Here we describe two methods for detecting ErbB3 protein in serum from patients who have undergone a clinical trial, utilizing two well-established methods in molecular biology-western blotting and ELISA, focusing on sample preparation and troubleshooting.