Double Trouble: Concomitant RB1 and BRCA2 Depletion Evokes Aggressive Phenotypes

In this issue of Clinical Cancer Research, Chakraborty and colleagues (1) build upon previous observations linking RB1 and BRCA2 defects to therapeutic bypass and poor outcome in prostate cancer (summarized in Fig. 1). While the encoded tumor suppressor proteins harbor distinct molecular functions, both genes are clustered in close chromosomal proximity. First identified by Cavenee and colleagues in retinoblastoma, RB1 resides at 13q14.2 (2), with BRCA2 just upstream (13q13.1). Using in vitro models of BRCA2 deletion, Chakraborty and colleagues demonstrated that BRCA2 loss bypasses the response to androgen depletion or androgen receptor antagonists. Subsequent RB1 knockdown in BRCA2-deficient cells increased in vitro migratory and invasive capacity, and induced epithelial-to-mesenchymal transition (EMT) markers, suggestive that coordinate BRCA2/RB1 loss shifts cells toward an alternate cell fate. The concept that RB1 loss can alter cell fate is well established in small-cell cancers and neuroendocrine prostate cancer, in which two copy, dual RB1 and TP53 tumor suppressor loss is a hallmark of disease (3). The impact of concomitant BRCA2/RB1 depletion in human disease was further considered using available clinical datasets; consistent with previous reports, depletion of either RB1 or BRCA2 was enriched in castration-resistant prostate cancer (4–6). Exciting new findings demonstrated that even single-copy BRCA2 loss is associated with aggressive disease, complementing previous reports showing that RB1 haploinsufficiency promotes therapeutic bypass and aggressive phenotypes (3, 7). Further analyses showed that codeletion (single or dual copy) of BRCA2 and RB1 associated with shorter disease- or progression-free survival, and was enriched in metastatic disease. Coloss of BRCA2/RB1 also associated with an increased fraction of the genome alteration, suggestive that dual tumor suppressor loss enhances genome instability. Finally, a 3-color FISH assay was developed to simultaneously monitor BRCA2, RB1, and 13q12 in cell lines and organoids, wherein single copy, concomitant loss of BRCA2 and RB1 resulted in sensitivity to PARP1/2 inhibitors. Combined, these findings advance the understanding of BRCA2 and RB1 dysfunction and identify dual tumor suppressor depletion (through single- or two copy loss) as promoting prostate cancer aggressiveness.

Observations were extended to assess the frequency and impact of coordinate BRCA2/RB1 deletion in other tumor types. Analyses of 10,820 pan-cancer in The Cancer Genome Atlas revealed that approximately 30% showed dual or single-copy codeletion of either RB1 or BRCA2. Interestingly, hetero- or homozygosity of either BRCA2 or RB1 associated with reduced overall survival, illustrating the impact of each tumor suppressor across cancer types. Beyond RB1 loss, RB pathway components alterations proved of importance; in Chakraborty and colleagues, BRCA2 mutations associated with increased risk of breast cancers wherein p16ink4a and cyclin D2 alterations occur frequently and may drive similar phenotypes (7). Further investigation into the frequency, cellular consequence, and clinical impact of coordinate BRCA2 and RB1 or RB pathway component loss will be an important next step toward determining the impact across tumor types.

A major advance of the study is the discovery that BRCA2 haploinsufficiency acts alone or in concert with RB1 loss to promote therapeutic bypass. These observations complement previous studies using in vitro models, xenografts, and analyses of human tissue which showed that single-copy RB1 loss or RB low expression is associated with poor outcome (8). This concept was underscored by mapping of the expanded E2F1 cistrome and transcriptome after targeted RB1 depletion, which is enriched in aggressive disease (5). Notably, it was shown that of prostate tumors with a single copy of RB1, only approximately half retained RB1 transcript expression, suggesting that mechanisms in addition to genomic loss regulate RB expression (8). Distinguishing between loss of RB1 and loss of RB protein function is of likely importance, as the consequence of RB inactivation is distinct from that of genomic loss of RB1 (5). Whether this BRCA2/RB1 coloss phenotype is sustained when BRCA2 loss is combined with RB inactivation requires further investigation. As such, determining the impact of single-copy BRCA2 and RB1 loss on BRCA2 and RB protein expression will assist in understanding the clinically relevant mechanisms by which dual tumor suppressor alterations impinge upon outcome.

Another major finding of the study was the observation that dual BRCA2/RB1 depletion associated with DNA damage and genomic instability. Consistent with a known role in homologous recombination, germline BRCA2 alterations are associated with increased prostate cancer risk (9), and both germline and somatic alterations of BRCA2 were associated with improved response to PARP1/2 inhibitors in the landmark TO-PARP trial (10). Although RB has indirect functions in controlling the expression of DNA repair factors through E2F1, and direct functions in regulating chromosome structure and stability (11), little is known with regard to the importance of these functions in prostate cancer. Here, elimination of BRCA2 in vitro increased DNA damage markers, as expected, but these markers were further enhanced by RB1 depletion. While sensitivity to PARP1/2 inhibitors was observed in the dual depletion models, less certain is whether this sensitivity is exacerbated in homozygous dual depletion, and the contribution of this outcome to disease progression. While it is tempting to speculate that dual tumor suppressor depletion may confer sensitivity to other DNA repair targeting strategies, this postulate awaits further testing. The observation in Chakraborty and colleagues that dual BRCA2/RB1 defects are associated with overall increased genome alteration heightens enthusiasm for understanding this process and discerning the role in cancer progression.

The mechanisms by which BRCA2 and RB1 individually contribute to the observed phenotypes will be important in future considerations. RB is a transcriptional coregulator that suppresses E2F activator transcription factors, and RB depletion results in a rewiring of E2F1 activity, that is, overrepresented castrate-resistant disease and associated with poor outcome (5). The transcriptional impact of two copy RB loss was also recently reported, and a pan-cancer signature of homozygous RB1 loss developed (12). While both signatures identify cell-cycle perturbation as an outcome of RB depletion, there is little evidence that RB-low or -deficient prostate adenocarcinomas exhibit a higher mitotic index in prostate cancer (5). Both previously reported signatures identify multiple processes of cancer relevance impacted by RB loss, including Myc, Notch, and Hedgehog signaling, along with metabolic pathways such as oxidative phosphorylation. These collective findings, including new observations herein regarding EMT, leave open to speculation which function(s) of RB most influence tumor suppression. The need to address this open question is enhanced by the recognition that RB plays cellular roles outside cell-cycle regulation and transcriptional control, including maintaining genome stability, facilitating DNA repair, and regulating chromatin structure (11). Similar open questions remain with regard to BRCA2 function, which has a major role in homologous recombination, but has also been identified as a direct modulator of DNA replication fork protection, telomere replication and protection, the processing of R-loops, chromosome segregation, and transcriptional regulation (13). Thus, the relative contribution of RB and BRCA2 to the transcriptional and cellular phenotypes identified and as related to clinical outcome remains opportunities for mechanistic investigation.

Irrespective of mechanism, the studies by Chakraborty and colleagues nominate dual BRCA2 and RB1 deficiency as a potentially distinct and targetable subtype of advanced prostate cancer that is deserving of further study. New observations in the study linking dual-deficient tumor suppressor prostate cancers as associated with EMT-like markers, increased genomic instability, and poor outcome represent significant advances that set the foundation for future analyses to assess applicability to other tumor types, and to develop effective strategies for treating “double trouble” cancers.

K.E. Knudsen is a paid consultant for CellCentric and Genentech, reports receiving commercial research grants from Celgene and Novartis, speakers bureau honoraria from Sanofi, Celgene, and Janssen, and holds ownership interest in Pfizer. No potential conflicts of interest were disclosed by the other author.

K.E. Knudsen was funded by the NIH (5R01CA217329-30 and 5P30CA056036-20).