Impact of Nuclear YAP1 Expression in Residual Cancer After Neoadjuvant Chemohormonal Therapy with Docetaxel for High-Risk Locali
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Matsuda et al. BMC Cancer (2020) 20:302 https://doi.org/10.1186/s12885-020-06844-y RESEARCH ARTICLE Open Access Impact of nuclear YAP1 expression in residual cancer after neoadjuvant chemohormonal therapy with docetaxel for high-risk localized prostate cancer Yoshinori Matsuda1, Shintaro Narita1*, Taketoshi Nara1, Huang Mingguo1, Hiromi Sato1, Atsushi Koizumi1, Sohei Kanda1, Kazuyuki Numakura1, Mitsuru Saito1, Takamitsu Inoue1, Yuko Hiroshima2, Hiroshi Nanjo2, Shigeru Satoh3, Norihiko Tsuchiya4 and Tomonori Habuchi1 Abstract Background: Although docetaxel-based chemohormonal therapy (CHT) is one of the standard treatments for castration-resistant prostate cancer (CRPC), pertinent biomarkers and precise mechanisms involved in the resistance for CHT for CRPC remain unknown. We investigated the relationship between chemohormonal resistance and the expression of steroid receptors and Hippo pathway proteins using a docetaxel-resistant prostate cancer (PCa) cell line and human PCa tissues in patients who underwent surgery with and without neoadjuvant therapy. Methods: A docetaxel-resistant subline (22Rv1-DR) was generated to assess Hippo pathway protein expression and the effect of YAP1 inhibition on cellular characteristics. A tissue microarray with 203 cores from 70 high-risk localized PCa tissues was performed to assess steroid receptor and Hippo pathway protein expressions. Results: Nuclear YAP (nYAP) expression was higher in 22RV-1-DR than in parental 22Rv-1 and YAP1 knockdown suppressed cell proliferation of 22Rv1-DR. Steroid receptor and Hippo pathway protein expressions varied among three different neoadjuvant groups, and nYAP1 expression was the highest in the CHT group. The patients with high nYAP in residual cancer after neoadjuvant CHT had a significantly higher biochemical recurrence (BCR) rate than those with low nYAP1. On multivariate analysis, the high nYAP1 was an independent prognostic factor for BCR. Conclusions: nYAP expression is a potential biomarker in high-risk patients treated with docetaxel-based CHT. Steroid receptors and Hippo pathway proteins may play a role in the chemohormonal resistance in advanced PCa. Keywords: YAP1, Residual cancer, Neoadjuvant chemohormonal therapy, Docetaxel, Prostate cancer, High-risk Background men in 92 countries and the leading cause of cancer Prostate cancer (PCa) is one of the most common can- deaths for men in 48 countries [2]. Although androgen cers and the major leading cause of cancer-related death deprivation therapy (ADT) is a mainstay treatment for in men worldwide [1]. A recent epidemiological study advanced PCa, eventually all patients develop conven- showed that PCa had the highest incidence of cancer for tional ADT-resistant cancer, known as castration- resistant prostate cancer (CRPC). After two large phase * Correspondence: [email protected] III randomized trials demonstrated that docetaxel-based 1Department of Urology, Akita University School of Medicine, 1-1-1 Hondo, chemotherapy improved the survival of patients with Akita 010-8543, Japan Full list of author information is available at the end of the article CRPC [3, 4], docetaxel-based chemotherapy is one of © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Matsuda et al. BMC Cancer (2020) 20:302 Page 2 of 14 the standard treatments for CRPC. However, a signifi- involved in docetaxel-resistance of prostate cancer [18, cant heterogeneity was found in the response of patients 19]. However, the impact of Hippo pathway proteins on to ADT and/or chemotherapy, and most patients be- ADT- and chemotherapy-resistant PCa progression re- come refractory due to the development of drug resist- mains unknown. In particular, no study has evaluated ance. Therefore, the identification of key regulators of the expression levels of the Hippo pathway proteins in resistance to both hormonal therapy and chemotherapy PCa patients with and without neoadjuvant treatment. in patients with advanced PCa is warranted. In this study, we investigated Hippo pathway protein Recent randomized phase III trials have shown that expression and the effect of YAP1 inhibition on cellular the combination therapy of ADT plus docetaxel for characteristics such as proliferation, apoptosis and the newly diagnosed metastatic hormone-naive PCa provides cell cycle in a docetaxel-resistant PCa cell line. More- significant survival benefits compared with ADT alone over, the tissue expression of candidate biomarkers, in- [5, 6]. However, whether the same extent of benefit is cluding Hippo pathway proteins and steroid receptors, obtained if these therapies are applied at an earlier stage, was assessed using human PCa tissues in patients who such as high-risk localized PCa, remains controversial. underwent RP with and without neoadjuvant therapy to We previously reported the outcomes of neoadjuvant identify tissue biomarkers in patients with high-risk PCa chemohormonal therapy (CHT) combined with ADT, treated with CHT and to explore novel targets for che- docetaxel, and estramustine phosphate, followed by rad- mohormonal resistance for advanced PCa. ical prostatectomy (RP) [7, 8] . In these studies, we found the limitation of the effect of CHT in patients Methods with high-risk localized PCa, therefore indicating the im- Cell lines portance of elucidating the molecular mechanisms Human PCa 22Rv1 cells were obtained from the underlying the resistance to docetaxel–CHT to over- American Type Cell Culture Collection (Manassas, VA, come resistance and improve the outcome for the treat- USA). The cells were authenticated with short-tandem ment of high-risk localized PCa. repeat analysis (Bio-Synthesis, Lewisville, TX, USA). The Numerous previous studies have hypothesized that docetaxel-resistant subline of 22Rv1 cell (22Rv1-DR), mechanisms and pathways are implicated in the develop- which has a characteristic of androgen- and ligand- ment of resistance to ADT and docetaxel in PCa [9–11]. independent growth [20, 21], was established in the Both androgen receptor (AR)-dependent and -independ- presence of increasing concentrations of docetaxel ent mechanisms were considered to be associated with (Sigma-Aldrich, St. Louis, MO, USA) up to the final resistance for PCa treatment [12]. Based on tissue bio- concentration of 5 nM, which is the IC concentration marker analyses, the expression of AR signaling, steroid 50 in our pilot study. Development of the drug-resistant cell synthesis pathway, intracellular signaling, stromal–epi- line took ≥4 months and further studies using sublines thelial interaction, hedgehog signaling, and angiogenesis cultured for ≥4 months that were based on the results of pathway were considered to be biomarkers for drug re- MDR1 expression in the cells were performed. The cells sistance and prognoses in patients treated with neoadju- were cultured in RPMI 1640 medium (Invitrogen, Carls- vant chemotherapy and CHT, followed by RP [13, 14]. bad, CA, USA) containing 10% fetal bovine serum (FBS) The Hippo pathway is known to regulate tissue in a 5% CO humidified incubator at 37 °C and passaged homeostasis, organ size, and tumorigenesis, and exerts a 2 for 3–4 days in a fresh medium to achieve approximately significant impact on cancer development by modulating 80% confluency. SiYAP1 (SI02662954) and negative con- cell proliferation, apoptosis, and stemness in response to trol siRNA (AllStars Negative Control siRNA) were pur- a wide range of extra- and intracellular signals, including chased from Qiagen (Valencia, CA). Transfections of cell–cell contact, cell polarity, mechanical cues, ligands siRNAs were performed by using Lipofectamine 3000 of G-protein-coupled receptors, and cellular energy sta- (Invitrogen) according to the manufacturer’s procedure. tus [15]. Moreover, increased nuclear localization and higher transcriptional activities of YAP/TAZ, main tran- scriptional regulators in the Hippo pathway, have been Cell proliferation assay observed in therapy-resistant malignancies [16]. With re- A non-radioactive MTT-based cell proliferation assay kit gard to the relationship between Hippo signaling and (Roche, Switzerland) was used based on the manufac- PCa, a recent study showed that YAP1 and AR are co- turer’s instructions. The proliferation assays were per- localized and interact with each other predominantly formed in triplicate. A total of 1.0 × 104 22Rv1 cells were within the cell nuclei by an androgen-dependent mech- seeded into each well of a 24-well plate and incubated anism in hormone-naive PCa and an androgen-