Non-canonical functions of the RB protein in cancer

Abstract

The canonical model of RB-mediated tumour suppression developed over the past 30 years is based on the regulation of E2F transcription factors to restrict cell cycle progression. Several additional functions have been proposed for RB, on the basis of which a non-canonical RB pathway can be described. Mechanistically, the non-canonical RB pathway promotes histone modification and regulates chromosome structure in a manner distinct from cell cycle regulation. These functions have implications for chemotherapy response and resistance to targeted anticancer agents. This Opinion offers a framework to guide future studies of RB in basic and clinical research.

Fig. 1 |. Canonical RB-E2F regulation and mechanisms of CDK resistance.

a | An illustration of our definition of canonical RB function is presented. In this model, growth factors signal the expression and activation of D-type cyclins. Cyclin D-cyclin-dependent kinase 4 (CDK4) or CDK6 and cyclin E-CDK2 act to hyperphosphorylate RB. Members of the E2F family of transcription factors and their dimeric partners (DPs) are released to activate the expression of genes that advance the cell cycle. This signalling pathway can be blocked by CDK inhibitor 2A (CDKN2A)-mediated inhibition of cyclin D, which is associated with CDK activity, b | RB can escape CDK hyperphosphorylation when its RxL motif is bound by protein phosphatase 1 (PP1) or when the RxL motif is acetylated or methylated, collectively depicted as RxL motif inhibition. This prevents substrate recognition and ensures retention of RB function. c | RB and transcription factor E2F1 can interact through molecular contacts that are distinct from RB and other E2F family members. In this interaction, CDK hyperphosphorylation of RB is unable to disrupt E2F1 binding. Similarly, ultraviolet light or osmotic shock can activate p38 MAPK to phosphorylate RB. Once phosphorylated, this stimulates E2F1 binding, even when RB is hyperphosphorylated by CDKs. d | RB-E2F complexes can recruit chromatin-modifying enzymes such as enhancer of zeste homologue 2 (EZH2) to mediate patterns of methylation of histone H3 lysine 27 (H3K27me3) at promoters. RB is removed from these locations by CDK hyperphosphorylation at the onset of S phase, but histone tail modification patterns are preserved during DNA replication, enabling stable transmission of the epigenetic state to the next cell generation. In the ensuing G1 phase, RB is dephosphorylated by PP1A and RB-E2F recruitment of histone methyltransferase activity reinforces patterns of modification in the ensuing cell cycle.

Fig. 2 |

2F family members and extensively throughout the genome at repetitive sequences and replication origins with transcription factor E2F1 (and likely other recruiting factors (RFs)) in a sequence-independent manner. b | The distribution of RB throughout the genome using RFs that are not sequence-specific leaves it poised to participate at sites of DNA breaks for non-homologous end joining (NHEJ) and homologous recombination (HR) repair in complex with X-ray repair cross-complementing protein 5 (XRCC5), XRCC6 or transcription activator BRG1. RB also recruits cohesin and condensin II complexes to replicating DNA and mitotic chromosomes for condensation and segregation. Lastly, RB directs histone deacetylation through the nucleosome-remodelling and deacetylase (NuRD) complex and histone H3 lysine 27 trimethylation (H3K27me3) using enhancer of zeste homologue 2 (EZH2) at repeats and enhancers. Ac, acetyl.

Fig. 3 |. Proposed model for RB1 loss in transdifferentiation and drug resistance.

a | RB1 gene mutation is a frequent event in acquired resistance to targeted therapies in which the initial adenocarcinoma transdifferentiates into a neuroendocrine cancer. b | Initially, the adenocarcinoma cells express RB, which binds to DNA regions via recruitment factors (RFs) such as transcription factor E2F1, and use enhancer of zeste homologue 2 (EZH2) to establish histone H3 lysine 27 trimethylation (H3K27me3) and repress repeats, including the repeat-like gene paternally expressed 10 (PEG10) and stem cell-related factors such as transcription factor SOX2. Following treatment with a targeted agent that inhibits a driver oncogene in the adenocarcinoma, cells that mutate RB1 are able to change H3K27me3 patterns and express SOX2 and PEG10 to establish a new cell identity that is no longer dependent on the therapeutic target. Treatment of resistant neuroendocrine tumours with EZH2 inhibitors presumably reorganizes H3K27me3 and gene expression patterns, returning cells to an epithelial state and restoring expression and sensitivity to inhibition of the original driver oncogene. These molecular events in adenocarcinoma to neuroendocrine transdifferentiation were established by studying prostate cancer, while only RB1 loss and SOX2 transcription have been presently implicated in transdifferentiation of epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma. AR, androgen receptor.

Fig. 4 |. Features of the canonical and non-canonical RB pathways.

a | Positive growth signals activate cyclin-dependent kinases (CDKs) to phosphorylate and inactivate RB, whereas negative growth regulators (DNA damage and transforming growth factor-β (TGFβ)) augment CDK inhibitors. Inactivation of RB in the canonical pathway leads to E2F target gene transcription. b | Non-canonical RB functions are best defined by their ambivalence to CDK regulation. Signals that likely activate non-canonical RB include stresses that activate p38 MAPK phosphorylation and DNA damage that stimulates P300-associated factor (PCAF)-dependent acetylation and SET domain-containing protein 8 (SET8; also known as KMT5A) or SET and MYND domain-containing protein (SMYD)-dependent methylation. With the aid of recruitment factors (RFs), often transcription factor E2F1, RB is capable of maintaining genome stability by localizing to sites of DNA breaks and stimulating non-homologous end joining or homologous recombination repair in complex with the DNA binding proteins X-ray repair cross-complementing protein 5 (XRCC5), XRCC6 or transcription activator BRG1. RB also ensures fidelity of DNA replication and chromosome condensation through direct interactions with condensin II complexes and the indirect influence of cohesin recruitment. Lastly, RB recruits enhancer of zeste homologue 3 (EZH2) to trimethylate histone H3 lysine 27 (H3K27me3) and inhibit enhancers to control lineage commitment and to silence expression of repetitive sequences. Importantly, this diverse grouping of functions is facilitated by the association of RB with the genome over a much larger scale than proximal promoters. DP, dimeric partner.