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  • Triple negative breast cancer TNBC accounts for of all diagn

    2020-08-28

    Triple-negative breast cancer (TNBC) accounts for 10–15% of all diagnosed breast cancers and has an aggressive clinical course due to a lack of therapeutic cellular targets [1]. Accumulating clinical evidence demonstrates that TNBC is biologically more aggressive and has a higher Ki-67 proliferation index with greater metastatic potential than other breast cancer subtypes [2,3]. Consensus toward a standard ther-apeutic strategy is lacking, and the overall five-year survival rate is less than 30% in TNBC patients who present with a distant recurrence [4].
    Heat-shock protein 90 (HSP90) is a ubiquitous molecular chaperone that is evolutionally conserved and highly abundant, comprising 1–2% of total cellular protein under normal physiological conditions [5].
    During cancer progression, this proportion increases by 2–10-fold in tumor G-418 [6]. HSP90 plays important roles in numerous biological functions and diverse processes including cell survival, proliferation, cancer progression and metastasis by regulating stability, maturation and the conformational changes of various proteins [7–9]. Elevated HSP90 levels are frequently observed in TNBC patients, which is asso-ciated with a higher risk of recurrence, distant metastasis and a poor prognosis [10]. HSP90 dysfunction with natural and synthetic in-hibitors attenuates cell proliferation, survival and cell dissemination in many cancer types via dysregulation of HSP90 client oncoproteins in-cluding HER2, EGFR, VEGF, JAK, STAT, and AKT [11–14].
    Of particular note, HSP90 is essential for the functional competence of STAT3 activity that governs the tumor microenvironment and cancer
    1 T-M. Cho and JY. Kim contributed equally to this work.
    Fig. 1. L80 suppresses TNBC cell viability in a dose- and time-dependent manner. (A) Chemical structures of deguelin and L80. (B) Changes in cellular morphology in MDA-MB-231, 4T1, BT549 and Hs578T cells after L80 (5 μM) treatment for 72 h as seen through phase contrast microscopy. (C) Effect of L80 on cell viability. Cells were treated with various concentrations of L80 (0.2–20 μM) for 48 h and 72 h. Cell viability was determined by MTS assay (*p < 0.05, versus DMSO control). The results are presented as mean ± SEM of at least three independent experiments and analyzed by two-way ANOVA followed by Bonferroni's post hoc test.
    progression [15]. The constitutive activation of STAT3 in TNBC is as-sociated with drug resistance to chemotherapy and a shorter survival period [16,17]. Blockage of the JAK/STAT axis elicits anti-angiogenic and -metastatic effects via suppression of the downstream signaling pathways involved [18–20]. A specific and direct interaction between HSP90 and STAT3 has been observed, which is a prerequisite for STAT3 interaction with JAK kinases, phosphorylation, dimerization, and nu-clear translocation of STAT3, contributing to tumor cell survival [15,21]. Therefore, HSP90 inhibition might serve to simultaneously 
    block both HSP90 function and STAT3 signaling.
    HSP90 consists of three distinct domains, an N-terminal ATP-binding domain, a middle domain, and a C-terminal dimerization do-main [9]. To date, the majority of HSP90 inhibitors in drug develop-ment target the N-terminal ATP-binding domain. Although several N-terminal HSP90 inhibitors are in clinical trials, none are currently available for routine cancer treatment [11,22]. The major impediment for N-terminal inhibitors is the induction of the heat shock response (HSR) and considerable upregulation of co-chaperones (HSP70 and
    HSP27) leading to the suppression of apoptosis via interference with key apoptotic-factors such as caspases, death receptors, bax and for-mation of the apoptosome [23,24]. In this context, further prospects in drug discovery of HSP90 inhibitors will need to focus on targeting the
    interaction of HSP90 with co-chaperone proteins (CDC37, HSP70 and HSF-1), including the C-terminal dimerization domain or downstream clients [22].
    We synthesized the C-ring truncated deguelin derivative L80 as a C-
    (caption on next page)
    Fig. 2. L80-induces apoptosis accompanied by caspase-3 activity. (A) MDA-MB-231 and 4T1 cells were treated with L80 (0–10 μM, 72 h) and early and late apoptosis assays with annexin V/PI staining were performed with flow cytometry. The percentages of the annexin V-positive cell populations have been quantified (right panel, *p < 0.05). (B–C) Effect of L80 on caspase-3 activity. Cells were pre-cultured with Z-VAD-fmk (20 μM, 1 h) before L80 treatment (0–10 μM, 72 h) and caspase-3 activity was analyzed by spectrophotometer. Data were analyzed by one-way ANOVA followed by Bonferroni's post hoc test, (**p < 0.01, DMSO vs L80; ##p < 0.01, L80 treatment alone vs combination treatment with Z-VAD-fmk and L80). (D) L80 does not affect apoptosis in normal cells in vitro. Normal human mammary epithelial MCF10A, normal murine mammary gland NMuMG cells or normal human embryonic kidney HEK293 cells were treated with L80 (5 μM) or deguelin (0.5 μM) for 72 h. Apoptosis assays with annexin V/PI staining were performed using flow cytometry, and the percentages of the annexin V-positive cells were quantified. The results are presented as mean ± SEM of at least three independent experiments. Data were analyzed by Student's t-test (**p < 0.01, DMSO vs deguelin (Deg); NS, not significant, DMSO vs L80).