Published On: Thu, Apr 18th, 2019

The chromodomain helicase CHD4 regulates ERBB2 signaling pathway and autophagy in ERBB2+ breast cancer cells [RESEARCH ARTICLE]


Breast cancer (BC) is considered a collection of diseases showing heterogeneity at molecular, histopathological and clinical level, which generates variable clinical courses and responses to treatments (Polyak, 2011). The genetic and molecular characterization of breast tumors has allowed the identification of five main subtypes according to the receptor status (estrogen, progesterone or ERBB2) (Goldhirsch et al., 2011). Among them, the ERBB2 overexpressing (ERBB2+) subtype is characterized by amplification or overexpression of the ERBB2 (ERBB2/Neu) oncogene and accounts for approximately 20–30% of all BCs (Yarden, 2001a). ERBB2 belongs to the human epidermal growth factor receptor (EGFR) family, which consists of four members (ERBB1/EGFR, ERBB2, ERBB3 and ERBB4). Of the four ERBB receptors, only ERBB2 has no known ligand and is subjected to an additional layer of regulation mediated by the molecular chaperone HSP90 (Castagnola et al., 2016; Bertelsen and Stang, 2014; Miyata et al., 2013). Several malignancies are associated with mutations or increased expression of members of the EGFR family, including lung, breast, stomach, colorectal, head and neck, thyroid, pancreatic carcinomas and glioblastoma (Yarden, 2001b; Li et al., 2018; Minuto et al., 2018; Sigismund et al., 2018; von Achenbach et al., 2018; Rodríguez-Antona et al., 2010). The ERBB receptors work as homo- or heterodimers able to engage different downstream signaling modules, such as Ras/Raf/MAPK and phosphatidylinositol 3-kinase (PI3K)/AKT pathways (Harari and Yarden, 2000; Carmona et al., 2016; Bagnato et al., 2017). In addition, ERBB2 overexpression correlates with increased progression through the cell cycle by affecting CDKN1A/p21WAF1 and CDKN1B/p27KIP1 (Carmona et al., 2016).

Trastuzumab (Tz) is an inhibitory monoclonal antibody that targets the extracellular domain of ERBB2 and is used as a front-line therapy for the treatment of ERBB2+ BCs. Tz downregulates the downstream PI3K/AKT and Ras/Raf/MEK/ERK1/2 signaling cascade, resulting in the impairment of cell proliferation (Yakes et al., 2002; Vu and Claret, 2012). Moreover, ERBB2 endocytic downregulation, cell cycle arrest in G1 phase and nuclear accumulation of the cell cycle inhibitor p27KIP1 have been reported (Valabrega et al., 2005; Nahta and Esteva, 2006; Le et al., 2005). Combinations of Tz with chemotherapeutic agents or other targeted inhibitors has reduced recurrence rates, improved outcome and prolonged the survival of patients; however, de novo and acquired resistance to Tz are still frequently observed (Nahta and Esteva, 2006; Lavaud and Andre, 2014; Di Modica et al., 2017).

The catabolic process of autophagy is a protein degradation process regulated by the mTOR-signaling pathway, which degrades cytoplasmic constituents within lysosomes (Yin et al., 2016). In cancer biology, autophagy has emerged as a resistance mechanism to multiple anticancer therapies such as kinase inhibitors or chemotherapy (Amaravadi et al., 2011). Protective autophagy might be induced in BC cells treated with anti-ERBB2 drugs such as Lapatinib or Tz, allowing cancer cells to survive (Chen et al., 2016; Vazquez-Martin et al., 2009). For these reasons, autophagy inhibitors are under intense investigations as novel anti-cancer agents (Amaravadi et al., 2011; Bortnik and Gorski, 2017). Recently, we demonstrated that the diterpene carnosic acid (CA) in combination with Tz impairs late autophagy, partially restoring Tz sensitivity in Tz-resistant cells (D’Alesio et al., 2017).

The chromatin remodeling helicase CHD4, a component of the nucleosome remodeling and deacetylases (NuRD) complex, has been recently identified as an essential regulator of BC growth in murine and patient derived xenograft (PDX) BCs (D’Alesio et al., 2016) and correlates with poor prognosis in cancers (Nio et al., 2015; Xia et al., 2017). In addition to its role in transcriptional regulation, CHD4 is also implicated in DNA damage response, cell cycle progression (O’Shaughnessy and Hendrich, 2013), cell stemness in a model of hepatocellular carcinoma (Nio et al., 2015) and in organogenesis and postnatal organ/tissue differentiation (Gómez-Del Arco et al., 2016). In a triple negative BC cell line, CHD4 depletion causes a significant reduction of cell proliferation and migration in vitro and a dramatic decrease of the tumor mass in vivo (D’Alesio et al., 2016). This inhibition was also found in luminal B and triple negative PDX models and in a transgenic mouse model (MMTV/NeuT) having the rat ERBB2 ortholog activated (D’Alesio et al., 2016). Moreover, CHD4 regulates BC cell cycle progression and its silencing determines the accumulation of cells in the G0 phase, a dramatic reduction of DNA synthesis, together with an upregulation of p21WAF1 (D’Alesio et al., 2016). Most importantly, the depletion of CHD4 in MCF10A cells, a human mammary epithelial cell line that lacks tumorigenic potential, did not affect cell proliferation and migration in vitro, suggesting that CHD4 targeting has the potential to become a novel therapeutic strategy to impair BC progression (D’Alesio et al., 2016).

Interestingly, evidence shows that the NuRD complex plays a role in the epigenetic regulation of autophagy. It has been demonstrated that repression of mTOR expression by SOX2 promotes cellular reprogramming and induction of autophagy through the recruitment of the NuRD complex (Wang et al., 2013). In addition, the methyltransferase EZH2 represses the expression of mTOR pathway-related genes via the NuRD complex component MTA2 (metastasis associated 1 family, member 2) (Wei et al., 2015).

In this work, we aimed at filling the gap of knowledge about the role of CHD4 in the specific regulation of the ERBB2-mediated signaling cascades and autophagy in ERBB2+ BC cells. We have found that CHD4 depletion impairs ERBB2 molecular pathways downregulating the phosphorylation status of pAKT and pERK. In addition, we demonstrated that CHD4 silencing impairs late stages of autophagy likely contributing to the impairment of BC cell proliferation. Lastly, we showed that CHD4 deprivation cooperates with Tz in zeroing ERBB2+ BC cell proliferation. Our work provides new insights on CHD4 as a potential target for the treatment of ERBB2+ BC to be used alone or in combination with traditional anticancer agents.

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