Tawna L Mangosh1,2, Magdalena M Grabowska1,2,3,4, Derek J Taylor1,2,4. 1. Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. 2. Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. 3. Department of Urology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. 4. Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Abstract
BACKGROUND: To ensure replicative immortality in cancer, telomeres must be maintained through activation of telomere maintenance mechanisms (TMMs) that are dependent on telomerase or the alternative lengthening of telomeres (ALT) pathway. Although TMM pathways have traditionally been considered to be mutually exclusive, ALT hallmarks have been identified in cancers defined as being telomerase-positive, supporting TMM coexistence. In castration-resistant prostate cancer (CRPC), in vitro models were thought to be universally dependent on telomerase as the primary TMM; however, CRPC models with androgen receptor (AR) loss demonstrate ALT hallmarks with limited telomerase activity and require ALT-associated PML bodies (APBs) for sustained telomere maintenance. The TMM coexistence in AR-negative CRPC is reliant on the ALT regulator protein, SLX4IP. METHODS: To identify the regions of SLX4IP responsible for the induction of APBs and telomere preservation in CRPC models, five 3xFLAG-tagged SLX4IP constructs were designed and stably introduced into parental C4-2B, DU145, and PC-3 cells. Once generated, these cell lines were interrogated for APB abundance and SLX4IP construct localization via immunofluorescence-fluorescence in situ hybridization (IF-FISH) and coimmunoprecipitation experiments for telomeric localization. Similarly, PC-3 cells with endogenous SLX4IP knockdown and SLX4IP construct introduction were interrogated for APB abundance, telomere length preservation, and senescent rescue. RESULTS: Here, we define the N-terminus of SLX4IP as being responsible for the promotion of the ALT-like phenotype of AR-negative CRPC models. Specifically, the N-terminus of SLX4IP was sufficient for promoting APB formation to a similar degree as full-length SLX4IP across CRPC cell lines. Additionally, APB promotion by the N-terminus of SLX4IP rescued telomere shortening and senescent induction triggered by SLX4IP knockdown in AR-negative CRPC cells. Moreover, APB formation and telomere maintenance were dependent on the ability of the N-terminus to direct SLX4IP localization at telomeres and APBs. CONCLUSIONS: These findings identify the role of the uncharacterized ALT regulator SLX4IP in the promotion of TMM coexistence to perpetuate replicative immortality in CRPC in vitro.
BACKGROUND: To ensure replicative immortality in cancer, telomeres must be maintained through activation of telomere maintenance mechanisms (TMMs) that are dependent on telomerase or the alternative lengthening of telomeres (ALT) pathway. Although TMM pathways have traditionally been considered to be mutually exclusive, ALT hallmarks have been identified in cancers defined as being telomerase-positive, supporting TMM coexistence. In castration-resistant prostate cancer (CRPC), in vitro models were thought to be universally dependent on telomerase as the primary TMM; however, CRPC models with androgen receptor (AR) loss demonstrate ALT hallmarks with limited telomerase activity and require ALT-associated PML bodies (APBs) for sustained telomere maintenance. The TMM coexistence in AR-negative CRPC is reliant on the ALT regulator protein, SLX4IP. METHODS: To identify the regions of SLX4IP responsible for the induction of APBs and telomere preservation in CRPC models, five 3xFLAG-tagged SLX4IP constructs were designed and stably introduced into parental C4-2B, DU145, and PC-3 cells. Once generated, these cell lines were interrogated for APB abundance and SLX4IP construct localization via immunofluorescence-fluorescence in situ hybridization (IF-FISH) and coimmunoprecipitation experiments for telomeric localization. Similarly, PC-3 cells with endogenous SLX4IP knockdown and SLX4IP construct introduction were interrogated for APB abundance, telomere length preservation, and senescent rescue. RESULTS: Here, we define the N-terminus of SLX4IP as being responsible for the promotion of the ALT-like phenotype of AR-negative CRPC models. Specifically, the N-terminus of SLX4IP was sufficient for promoting APB formation to a similar degree as full-length SLX4IP across CRPC cell lines. Additionally, APB promotion by the N-terminus of SLX4IP rescued telomere shortening and senescent induction triggered by SLX4IP knockdown in AR-negative CRPC cells. Moreover, APB formation and telomere maintenance were dependent on the ability of the N-terminus to direct SLX4IP localization at telomeres and APBs. CONCLUSIONS: These findings identify the role of the uncharacterized ALT regulator SLX4IP in the promotion of TMM coexistence to perpetuate replicative immortality in CRPC in vitro.
Authors: Jian Hu; Soyoon Sarah Hwang; Marc Liesa; Boyi Gan; Ergun Sahin; Mariela Jaskelioff; Zhihu Ding; Haoqiang Ying; Adam T Boutin; Hailei Zhang; Shawn Johnson; Elena Ivanova; Maria Kost-Alimova; Alexei Protopopov; Yaoqi Alan Wang; Orian S Shirihai; Lynda Chin; Ronald A DePinho Journal: Cell Date: 2012-02-17 Impact factor: 41.582
Authors: Adrie van Bokhoven; Marileila Varella-Garcia; Christopher Korch; Widya U Johannes; E Erin Smith; Heidi L Miller; Steven K Nordeen; Gary J Miller; M Scott Lucia Journal: Prostate Date: 2003-11-01 Impact factor: 4.104
Authors: Himisha Beltran; Scott Tomlins; Ana Aparicio; Vivek Arora; David Rickman; Gustavo Ayala; Jiaoti Huang; Lawrence True; Martin E Gleave; Howard Soule; Christopher Logothetis; Mark A Rubin Journal: Clin Cancer Res Date: 2014-04-11 Impact factor: 12.531