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Retinoblastoma Protein 1: History Edit
Subjects: Oncology

Retinoblastoma protein 1 (RB1) is encoded by a tumor suppressor gene that was discovered more than 30 years ago. Almost all mitogenic signals promote cell cycle progression by braking on the function of RB1 protein through mono- and subsequent hyper-phosphorylation mediated by cyclin-CDK complexes.

  • RB1
  • E2F
  • chromatin instability
  • synthetic lethality
  • collateral lethality

1. Introduction

The discovery of RB1 gene had paved an avenue toward understanding the complicated functions of the human genome in preventing cells from carcinogenesis [1,2]. RB1 gene was thought to be mechanistically involved in the initiation of retinoblastoma as its inactivation following genetic mutation or deletion was prevalently identified in the pedigrees of hereditary cases [3]. This notion was later experimentally proven by the induction of retinoblastoma in mice following the ablation of multiple Rb family members [4]. Together with the presence of other cancer driving mutations in genes such as Trp53, RB1 loss can provoke carcinogenesis from various types of tissue. This suggests that RB1 may in fact be involved in the initiation and/or progression of previously unexpected varieties of cancer [5]. Of note, RB1 inactivation is detected in close to 90% of sporadic small cell lung cancers (SCLC), suggesting a contribution to their initiation, while its germ line mutation also often predisposes to SCLC [6]. Reportedly, RB1 inactivation occurs in 20~40% of osteosarcoma cases, yet it is thought to contribute to tumor progression based on pathological examination and linkage with unfavorable outcome [7,8]. RB1 reconstitution in RB1-deficient osteosarcoma-derived cells does not always completely attenuate their malignant phenotypes, presumably due to coexisting functional aberration in Trp53 tumor suppressor or other driver mutations [9,10]. These findings also indicate that RB1 may exert varied functions depending on cancer tissue type and timing during the course of tumor development.
Similar to osteosarcoma, RB1 is suggested to be inactivated during their progression in a majority of cancer types [7]. One of the best examples is prostate cancer. In primary/non-metastatic prostate cancers, RB1 deletion is identified with less than 10% prevalence, but this rises to higher than 30% in those attaining metastatic and/or castration-resistant phenotypes [11]. In addition to prostate cancer, many common types of cancers, including non-small cell lung cancer (NSCLC), breast cancer, bladder cancer, chronic myelogenous leukemia (CML), high-grade glioma, esophageal cancer, head and neck cancer and sarcoma, are thought to gain RB1 deficiency during their malignant progression [7].
The roles of RB1 in suppressing malignant progression of prostate cancer may not be solely explained by its primary function to constrain cell cycle progression. For example, RB1 controls the transcription of androgen receptor (AR) through the E2F family of transcription factors, hence its inactivation could enhance the refractoriness to endocrine therapy [12]. Moreover, the depletion of RB1 from RB1-proficient prostate cancer cells promoted gain of stem cell-like properties through increased expression of interleukin-6 (IL-6) and lysyl oxidase (LOX) [13]. Similarly, RB1 inactivation in breast cancers enhances stem cell-like behaviors and malignant progression through IL-6 and following activation of signal transducer and activator of transcription 3 (STAT3) activation [14,15,16,17,18]. RB1 loss in breast cancer cells activates fatty acid oxidation (FAO) and induces following Jun kinase (JNK) activation. Activated JNK then stimulates IL-6 production leading to gain of undifferentiated characters which are mediated by cell-autonomous STAT3 activation [14]. Later, the enhanced FAO following RB1 loss was explained by AMP-activated protein kinase (AMPK) activation and subsequent phosphorylation (downregulation) of acetyl-CoA carboxylase (ACC) [16]. Accordingly, the blockade of IL-6 by neutralizing antibody suppressed breast cancer progression is induced following RB1 inactivation [14]. Loss of Trp53 in mice generates well differentiated soft tissue sarcoma with low prevalence; additional loss of Rb1 converts this to undifferentiated pleomorphic type [17]. An increase in lineage plasticity has been observed in mouse Trp53-null osteosarcoma cells following Rb1 deletion [19]. Identification of molecules mediating RB1 functions in controlling undifferentiated characters of tumor cells may endow us new tools to target cancer stem cells [18]. Taken together, in addition to targeting aberrant cell cycle progression, functional suppression of the proteins aberrantly expressed as a result of RB1 inactivation may also be beneficial in cancer treatment.

2. Targeting Gene Products Upregulated Following RB1 Inactivation

The RB1/E2Fs/DPs complex primarily suppresses target gene transcription by recruiting histone deacetylase (HDAC). Loss of RB1 function liberates its most important counterpart, E2Fs, and these primarily transactivate target genes by recruiting histone acetylase (HAT). The target genes of the “transactivating” E2Fs (E2F1, E2F2 and E2F3a) contain a number of genes enrolled in cell cycle progression, DNA synthesis and replication, DNA damage repair and apoptosis [20]. In line with this, the deletion of E2f1 suppressed pituitary and thyroid tumorigenesis and prolonged the life span in Rb1-heterozygous mice [21]. Likewise, the loss of E2f2 or E2f3 in MMTV-Myc transgenic mice resulted in the cessation of mammary carcinogenesis [22]. E2F1 is frequently overexpressed in metastatic melanoma, and inhibition of E2F1 induced apoptosis and cellular senescence [23]. In specific types of cancer, higher expression of some of E2F family members correlates with advanced stage and poor prognosis [24]. Therefore, targeting E2Fs may yield certain benefits in cancer therapy. The E2F inhibitor HML006474 was designed based on a virtual screening of compounds that possibly inhibit DNA binding of the E2F4/DP2 complex. The E2F4-null cells were less sensitive to this compound compared to wild type, which supports the specificity of its action [25]. However, to date, no small molecules specifically targeting any of transactivating E2Fs have been described nor tested in clinical trials.
As E2Fs target a wide variety of genes that could promote or suppress tumor development, it is much safer to consider strategies to target individual E2F target. As numerous review articles have already expounded on this point, this article picks up only a few. In particular, although cyclin E and cyclin dependent kinase 2 (CDK2) are representative targets of E2Fs during G1/S, and inhibitors to CDK2 are undergoing clinical trials [26], the efficacy of these agents and their dependency on the presence of intact RB1 remains a scantly addressed topic.
E2Fs target numerous genes involved in replication control, nucleotide synthesis, DNA damage response/repair, chromatin structure and mitotic progression. RB1 inactivation induces chromosomal instability (CIN), at least partially, by upregulating spindle assembly checkpoint protein mitotic arrest deficient 2 (MAD2). Overexpressed MAD2 may contribute to induced aneuploidy and stabilization of erroneous attachment of mitotic spindles [27,28]. Dysregulated expression of MAD2 is correlated to cancer progression and poor prognosis [29]. A small molecule, M2I-1, targeting MAD2-CDC20 interaction has been developed [30]. A report suggested that M2I-1 sensitizes cancer cells to anti-mitotic reagents [31] but, thus far, no report has tested its efficacy in RB1-deficient context. Many of mitotic kinases are upregulated following E2F dysregulation and inhibition of some of them has been indicated to be synthetically lethal with RB1 inactivation (see below).
Besides MAD2, numerous possibly druggable molecules have been indicated to be involved in the RB1 loss-induced CIN [32]. Aurora A and polo-like kinase 1 (PLK1) are upregulated following RB1 depletion, which may lead to aneuploidy or other mitotic abnormalities, further causing malignant progression [33]. Synthetic inhibitors to these kinases are now in clinical trials (Table 1). Mis-localization of CAPD3/condensin II has been proposed to mediate centromere dysfunction induced by RB1 loss [34]. However, synthetic inhibitors to this molecule are under development and it is not clear whether such inhibitors could exhibit anti-tumor activity.
Table 1. Genes whose inactivation exerts synthetic lethality in the presence of RB1 deficiency, and representative inhibitors to the gene products and phase of clinical trials. ND: no data. The information was obtained by searching online accessible catalogues provided by Selleck, Santa Cruz and Abcam.
Gene Inhibitor Phase of Clinical Trial
Aurora A, B Alisertib III
Tozasertib II
Barasertib I
MNL8054 I
Danusertib II
AT9283 II
KW-2449 I
SNS-341 I
ENMD-2076 II
BI-847325 I
PLK1 Volasertib III
Rigosertib III
BI2536 II
CHK1 AZD7762 I
MK-8779 II
PF-477736 I
CDC25A, B K-252a ND
NSC663284 ND
NSC95397 ND
WEE1 Adabosertib II
TAF1 CeMMEC-1, 13 ND
PARP Olaparib FDA-approved
Veliparib III
Rucapatib III
Iniparib III
SYK Fostamatinib III
R406 I
In addition to mitotic catastrophe, replicative catastrophe induced by abnormally enhanced cell cycle progression induced by gain of function by E2Fs could provide a certain vulnerability especially upon chemotherapy or radiation therapy [35]. The observation that RB1 inactivation in thyroid calcitonin-producing (C) cells or fibroblastic cells induced DNA damage response followed by senescence supports this speculation [36].
As alluded to the introduction, mostly in an E2F-dependent manner, RB1 status affects the milieu surrounding tumor cells including chemokine/cytokine secretion, extracellular matrix, immune cells or remote organs to be metastasized. The molecules mediating such functions of RB1 could serve as good therapeutic targets [37,38]. C-C motif chemokine 2 (CCL2) appeared to be secreted from RB1-deficient breast cancer cells, thereby such tumor cells recruit immunosuppressive cells including myeloid-derived suppressive cells (MDSC), regulatory T cells (Treg) or macrophages, allowing malignant progression following RB1 inactivation. Moreover, blockade of CCL2 signaling in host mice by genetically deleting CCR2, encoding the receptor for CCL2, almost completely abrogated mammary carcinogenesis from MMTV-cre; Rb1flox/flox mice [14,38]. RB1 inactivation has been linked to upregulation of PD-L1 expression through direct interaction with NF-κB protein p65. Surprisingly, a particular phosphorylated form of RB1 inactivates p65 and therefore attenuates expression of PD-L1 and cIAPs. The peptide mimicking this phosphorylated form of RB1 improved the therapeutic efficacy in radiotherapy by suppressing radiation-induced PD-L1 expression [39]. These findings make clear contrast with the enhanced immunogenicity induced by locking RB1 in unphosphorylated form by the treatment with CDK4/6 inhibitor [40], and suggest that RB1 status might affect the efficacy of therapy by immune checkpoint inhibitors (ICIs).
A while ago, an unexpected twist arose from C. elegans studies that indicated lin-35, an RB1 orthologue, might be placed upstream of Ras signaling during vulval development [41,42]. In cultured cells, including embryonic fibroblasts and thyroid C cells, K-Ras and N-Ras are activated 5~10 times greater following RB1 loss, and this may promote or suppress tumor development in a highly context-dependent manner [36,43,44,45,46,47]. It has been estimated that mutation of K-Ras or N-Ras in the context of RB1 loss exerts a mitogenic effect that is 50~60 times greater than that of corresponding wild type Ras in the presence of RB1. Mechanistically, the RB1-Ras nexus has been explained by farnesylation and geranylgeranylation to anchor cytosolic Ras proteins to lipid bilayers, which are innervated by E2Fs and SREBPs [36,48], thus this axis is pharmacologically targetable. These findings led researchers to address RB1 functions in cholesterol and then lipid metabolism. ELOVL fatty acid elongase 6 (ELOVL6) and fatty acid desaturase 1 (SCD1) have been linked to RB1 via E2Fs and SREBPs, which explained the profound impact of RB1 loss on the lipidomic landscape [49]. The oleic acid, one of the immediate products of ELOVL6 and SCD1, appeared to promote mammary carcinogenesis by stabilizing c-Myc through GPR40 which is a receptor for oleic acid, providing a good rationale to develop antagonists to this receptor to prevent breast cancer associated with high fat diet in puberty [50].
Although the dependence on E2Fs is still unclear, human retinoblastoma cells highly express a proto-oncogene spleen tyrosine kinase (SYK) and depend on its expression for survival. Treatment of genetic retinoblastoma model with synthetic SYK inhibitor resulted in dramatic suppression of disease progression and prolongation of survival [51]. Furthermore, in retinoblastoma cells, ubiquitin-like, containing PFD and RING finger domains 1 (UHRF1) and helicase, lymphoid specific (HELLS) are overexpressed, which is linked to epigenetic activation of SYK [52,53].

3. Partners Other Than E2Fs Liberated upon RB1 Loss

More than 300 proteins have been indicated to directly bind to RB1 [48]. Varied mono-phosphorylation status of RB1 (possibly 14 variations) may increase the complexity of the relationship with specific binding partners [54]. As RB1 is typically not abundantly expressed in tissues, RB1 may carefully select partners depending on the cellular context. A number of chromatin modifiers, cyclins and signaling molecules, such as apoptosis signal-regulating kinase 1 (ASK1) equipped with LxCxE motif, bind to RB1 at the site also targeted by a number of oncogenic virus products carrying LxCxE motif including adenovirus E1A, simian virus 40 large T antigen and human papilloma virus E7 [55]. Such chromatin modifiers include attractive targets of cancer therapy. Notably, 5-azacytidine targets DNMT1 and has been used for the treatment of myelodysplastic syndrome [56]. Similarly, the utility of pan-HDAC inhibitors has long been evaluated in a variety of cancers [57]. The Suv39H1 inhibitor chaetocin exhibited significant therapeutic efficacy against acute myeloid leukemia when combined with other epigenetic drugs that include suberoylanilide hydroxamic acid (HDAC inhibitor) and JQ-1 (bromodomain inhibitor) [58]. However, how the loss of RB1 impacts the efficacies and sensitivities of these molecules is not well studied. One notable exception is TAI-1, which targets Hec-1 and exhibits synergy with multiple chemotherapeutic agents in the treatment of leukemia, breast and liver cancer. In these settings, the status of RB1 and Trp53 influenced the sensitivity to TAI-1 [59].
SKP2 directly binds to RB1 [60], whereupon RB1 loss liberates SKP2 to promote the proteasomal degradation of p27KIP. SKP2 was initially identified as a cyclin A binding protein and later found to serve as a component of the SCFSKP2 E3 ubiquitin ligase complex targeting various substrates containing many of cell cycle regulators, including p27KIP. Additional loss of Skp2 loci appeared to increase the degree of apoptosis in Rb1-nullizygous mouse embryonic fibroblasts (MEFs) in an E2F-dependent manner. Similarly, Skp2 loss increased apoptotic cells in Rb1-deficient pituitary tumor developed in mice, also in an E2F-dependent manner [61]. Loss of SKP2 stabilizes p27KIP and simultaneously exposes cyclin A to p27KIP. Additionally, the loss of SKP2 lessens cyclin A activity to attenuate E2F1 function. Consequently, overactivated E2F1 behaves more proapoptotic rather than proliferative in the absence of RB1 [61].
Deficiency in terminal differentiation following RB1 inactivation would be another possible target in cancer treatment. Many of the tissue-specific transcription factors, including MYOD, CCAAT enhancer binding protein (C/EBP), glucocorticoid receptor (GR), GATA-binding factor 1 (GATA-1), PU-1, core-binding factor alpha 1 (CBFA-1), pancreas duodenum homeobox 1 (PDX1), runt-related transcription factor 2 (RUNX2) and nuclear factor of IL-6 expression (NF-IL6), directly or indirectly collaborate with RB1 to determine lineage specificity and to induce terminal differentiation [62]. As such, the loss of RB1 promotes undifferentiated behaviors of cancer cells, though the extent to which tissue-specific transcription factors also contribute to this phenotype is still unclear. Added to this, the functional recovery of tissue-specific transcription factors by pharmacological approaches may be difficult.
Yet how the loss of RB1 promotes undifferentiated behaviors of cancer cells might be at least partially explained by the pivotal roles of RB1 in controlling pluripotency factors including octamer-binding transcription factor 4 (OCT4) and SRY-box 2 (SOX2), by directly binding to the regulatory elements for these genes [63]. This study has been done in the context of inducible pluripotent stem cells (iPSs). Additionally, as abovementioned, IL-6 and LOX have been proposed to explain the effect of RB1 loss on the malignant progression of prostate and breast cancer [13,14]. Interference to these machineries may enable cancer therapy by inducing spontaneous differentiation.

This entry is adapted from the peer-reviewed paper 10.3390/cancers13153737