Functional Loss of ATRX and TERC Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells

Abstract

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRX KO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRX KO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRX KO TERC mut cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance.Implications: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.

Novel therapeutic strategies targeting telomere maintenance mechanisms in high-risk neuroblastoma

“…Further confirming the role of ATRX as an ALT repressor, ATRX knockdown has been shown to induce ALT activity in cells of mesenchymal origin [30]. However, ATRX depletion does not promote ALT activity in all cell types [31,32] suggesting that ATRX LoF alone is not sufficient to induce ALT and that additional, as yet unidentified mechanisms are also needed. Reinforcing the notion that ALT arises as a result a combination of ATRX loss and other factors, it has recently been shown that during the immortalisation process, ATRX loss results in a progressive chromatin de-compaction and a gradual induction of telomere replication dysfunction which triggers an adaptive response eventually resulting in ALT activation [33].…”

Cellular and Molecular Progression of Prostate Cancer: Models for Basic and Preclinical Research

“…Low-grade and high-grade prostate intraepithelial neoplasia (PIN) lesions develop from normal prostate epithelium through the loss of phosphatase and the tensin homolog (PTEN) , NK3 Homeobox 1 ( NKX3.1) , overexpression of MYC proto-oncogene, B-cell lymphoma 2 ( BCL-2), and the glutathione S-transferase pi 1 gene ( GSTP1), accompanied with Speckle Type BTB/POZ Protein ( SPOP) mutation and Transmembrane Serine Protease 2- ETS-related gene ( TMPRSS2-ERG) fusion [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Further loss of the retinoblastoma protein ( RB1) , along with telomerase activation and frequent Forkhead Box A1 ( FOXA1) mutation, leads to the development of prostate adenocarcinoma from the advanced PIN lesion [ 37 , 38 , 39 , 40 , 41 , 42 , 43 ]. Further molecular aberrations including the loss of SMAD Family Member 4 ( SMAD4) , AR corepressors, mutations in AR, FOXA1, BRCA1/2, ATM, ATR, and RAD51 accompanied with the gain of function of the AR coactivator, CXCL12, CXCR4, RANK-RANKL, EMT, BAI1 , and EZH2 lead to the development of metastatic prostate cancer [ 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ].…”

Alternative Lengthening of Telomeres and Chromatin Status

“…Recently, some authors succeeded in switching telomerase-positive cancer cell lines into ALT-active ones [63]. The study of the epigenetic modification of telomeres, before and after the switch, could be useful for elucidating the real chromatin modifications associated with ALT.…”