Downregulation of SIAH2, an ubiquitin E3 ligase, is associated with resistance to endocrine therapy in breast cancer

Abstract

International audienceIn our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance. SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression. SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; = 0.05) and progression-free survival (PFS) (HR = 0.79; = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells ( = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF. In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF

Decreased expression of EZH2 is associated with upregulation of ER and favorable outcome to tamoxifen in advanced breast cancer

“…To perform E ZH2 knockdown experim ents, small interfering RNA (siRNA ) targeting E ZH2 mRNA (Qiagen, Venlo, The Netherlands) was used according recommendations and described previously [17]. Two different siRNA duplexes for EZH2 were pooled with target sequences: r(CCA UGUUUACAA CUAUCAA)dTdT (sense) and r(UU GAUA GUUGUAAACA UGG)dTdT (antisens e) for the first duplex and r(GCAAAUUCUCGGUGUCAAA )dTdT (sens e) and r(UUUGA CACCGA GAAUUUGC)dTdT (antisense) for the second duplex.…”

“…EZH2 prot ein expression was visualized with a diaminobenzidine staining reaction. Western blotting of protein samples were performed as described previously [17]. Antibodies against EZH2 …”

A genome-wide association study identifies a genetic variant in the SIAH2 locus associated with hormonal receptor-positive breast cancer in Japanese

“…Previous reports implicated possible roles of SIAH2 in breast carcinogenesis and described that SIAH2 expression was highly associated with estrogen receptor levels. 9,[27][28][29] In addition, SIAH2 protein was indicated to have an essential role in the hypoxic response by regulating the hypoxia-inducible factor-a. 30 Moreover, SIAH2 was known to induce ubiquitin-mediated degradation of many substrates, including proteins involved in transcriptional regulation (POU2AF1, PML and NCOR1), a cell surface receptor (DCC) and an anti-apoptotic protein (BAG1).…”

“…6 In Japan, breast cancer is the most common cancer among women and its incidence has been doubled in both preand postmenopausal women in the last 20 years, mainly as an estrogen receptor-positive subgroup. 7 Although hormone therapy and radiotherapy are effective, cancer cells often become resistant to these treatments; nearly half of estrogen receptor-positive breast cancer patients at an advanced stage suffer from recurrence [8][9][10] and only onethird of hormonal receptor-positive (HRP) patients with metastatic disease respond to radiotherapy. 11 Therefore, new therapeutic options for the disease are eagerly awaited.…”

SIAH1 (siah E3 ubiquitin protein ligase 1)

“…Numerous substrates targeted for degradation by Siah proteins have been reported; Synphilin-1 (Nagano et al, 2003), DCC (Hu et al, 1997), N-CoR (Zhang et al, 1998), BOB1/OBF1 (Boehm et al, 2001, Tiedt et al, 2001), c-Myb (Tanikawa et al, 2000), Kid (Germani et al, 2000) and CtIP (Germani et al, 2003). Siah1 expression is upregulated by p53, revealing a link between genotoxic injury and destruction of β-catenin and reduced T-cell factor/lymphoid enhancer factor (Tcf/Lef) activity (Liu et al, 2001;Jansen et al, 2009). Recent paper showed Siah1 expression facilitates ubiquitination and degradation of the tumor suppressor HIPK2 that is a key regulator of the apoptotic programme induced by DNA damage (Winter et al, 2008).…”