CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.

Structural mechanisms of DREAM complex assembly and regulation.

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.

Clonal evidence for the development of neuroblastoma with extensive copy-neutral loss of heterozygosity arising in a mature teratoma.

Mature teratomas are usually benign tumors that rarely undergo malignant transformation. We report an advanced neuroblastoma arising in a mature teratoma of the ovary. Whole-exome sequencing identified extensive copy-neutral loss of heterozygosity (LOH) in both neuroblastoma and teratoma elements, suggesting that the neuroblastoma evolved from the teratoma. In addition, several truncating germline heterozygous variants in tumor suppressor genes, including RBL2 and FBXW12, became homozygous as a result of LOH. Collectively, we speculate that extensive LOH in teratoma cells may force heterozygous germline variants to become homozygous, which, in turn, may contribute to the development of neuroblastoma with the acquisition of additional chromosomal changes.

RBL2 bi-allelic truncating variants cause severe motor and cognitive impairment without evidence for abnormalities in DNA methylation or telomeric function.

RBL2/p130, a member of the retinoblastoma family of proteins, is a key regulator of cell division and propagates irreversible senescence. RBL2/p130 is also involved in neuronal differentiation and survival, and eliminating Rbl2 in certain mouse strains leads to embryonic lethality accompanied by an abnormal central nervous system (CNS) phenotype. Conflicting reports exist regarding a role of RBL2/p130 in transcriptional regulation of DNA methyltransferases (DNMTs), as well as the control of telomere length. Here we describe the phenotype of three patients carrying bi-allelic RBL2-truncating variants. All presented with infantile hypotonia, severe developmental delay and microcephaly. Malignancies were not reported in carriers or patients. Previous studies carried out on mice and human cultured cells, associated RBL2 loss to DNA methylation and telomere length dysregulation. Here, we investigated whether patient cells lacking RBL2 display related abnormalities. The study of primary patient fibroblasts did not detect abnormalities in expression of DNMTs. Furthermore, methylation levels of whole genome DNA, and specifically of pericentromeric repeats and subtelomeric regions, were unperturbed. RBL2-null fibroblasts show no evidence for abnormal elongation by telomeric recombination. Finally, gradual telomere shortening, and normal onset of senescence were observed following continuous culturing of RBL2-mutated fibroblasts. Thus, this study resolves uncertainties regarding a potential non-redundant role for RBL2 in DNA methylation and telomere length regulation, and indicates that loss of function variants in RBL2 cause a severe autosomal recessive neurodevelopmental disorder in humans.

Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells.

The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.

RB, p130 and p107 differentially repress G1/S and G2/M genes after p53 activation.

Cell cycle gene expression occurs in two waves. The G1/S genes encode factors required for DNA synthesis and the G2/M genes contribute to mitosis. The Retinoblastoma protein (RB) and DREAM complex (DP, RB-like, E2F4 and MuvB) cooperate to repress all cell cycle genes during G1 and inhibit entry into the cell cycle. DNA damage activates p53 leading to increased levels of p21 and inhibition of cell cycle progression. Whether the G1/S and G2/M genes are differentially repressed by RB and the RB-like proteins p130 and p107 in response to DNA damage is not known. We performed gene expression profiling of primary human fibroblasts upon DNA damage and assessed the effects on G1/S and G2/M genes. Upon p53 activation, p130 and RB cooperated to repress the G1/S genes. In addition, in the absence of RB and p130, p107 contributed to repression of G1/S genes. In contrast, G2/M genes were repressed by p130 and p107 after p53 activation. Furthermore, repression of G2/M genes by p107 and p130 led to reduced entry into mitosis. Our data demonstrates specific roles for RB, p130-DREAM, and p107-DREAM in p53 and p21 mediated repression of cell cycle genes.

The RB gene family controls the maturation state of the EndoC-ßH2 human pancreatic ß-cells.

The functional maturation of human pancreatic ß-cells remains poorly understood. EndoC-ßH2 is a human ß-cell line with a reversible immortalized phenotype. Removal of the two oncogenes, SV40LT and hTERT introduced for its propagation, stops proliferation, triggers cell size increase and senescence, promotes mitochondrial activity and amplifies several ß-cell traits and functions. Overall, these events recapitulate several aspects of functional ß-cell maturation. We report here that selective depletion of SV40LT, but not of hTERT, is sufficient to revert EndoC-ßH2 immortalization. SV40LT inhibits the activity of the RB family members and of P53. In EndoC-ßH2 cells, the knock-down of RB itself, and, to a lesser extent, of its relative P130, precludes most events triggered by SV40LT depletion. In contrast, the knock-down of P53 does not prevent reversion of immortalization. Thus, an increase in RB and P130 activity, but not in P53 activity, is required for functional maturation of EndoC-ßH2 cells upon SV40LT-depletion. In addition, RB and/or P130 depletion in SV40LT-expressing EndoC-ßH2 cells decreases cell size, stimulates proliferation, and decreases the expression of key ß-cell genes. Thus, despite SV40LT expression, EndoC-ßH2 cells have a residual RB activity, which when suppressed reverts them to a more immature phenotype. These results show that the expression and activity levels of RB family members, especially RB itself, regulate the maturation state of EndoC-ßH2 cells.

Rb suppresses human cone-precursor-derived retinoblastoma tumours.

Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

Dynamic site-specific recruitment of RBP2 by pocket protein p130 modulates H3K4 methylation on E2F-responsive promoters.

The Histone 3 lysine 4 methylation (H3K4me3) mark closely correlates with active transcription. E2F-responsive promoters display dynamic changes in H3K4 methylation during the course of cell cycle progression. However, how and when these marks are reset, is not known. Here we show that the retinoblastoma binding protein RBP2/KDM5A, capable of removing tri-methylation marks on H3K4, associates with the E2F4 transcription factor via the pocket protein-p130-in a cell-cycle-stage specific manner. The association of RBP2 with p130 is LxCxE motif dependent. RNAi experiments reveal that p130 recruits RBP2 to E2F-responsive promoters in early G1 phase to bring about H3K4 demethylation and gene repression. A point mutation in LxCxE motif of RBP2 renders it incapable of p130-interaction and hence, repression of E2F-regulated gene promoters. We also examine how RBP2 may be recruited to non-E2F responsive promoters. Our studies provide insight into how the chromatin landscape needs to be adjusted rapidly and periodically during cell-cycle progression, concomitantly with temporal transcription, to bring about expression/repression of specific gene sets.

Heat shock protein 27 promotes cell cycle progression by down-regulating E2F transcription factor 4 and retinoblastoma family protein p130.

Heat shock protein 27 (HSP27) protects cells under stress. Here, we demonstrate that HSP27 also promotes cell cycle progression of MRC-5 human lung fibroblast cells. Serum starvation for 24 h induced G1 arrest in these cells, and upon serum refeeding, the cells initiated cell cycle progression accompanied by an increase in HSP27 protein levels. HSP27 levels peaked at 12 h, and transcriptional up-regulation of six G2/M-related genes (CCNA2, CCNB1, CCNB2, CDC25C, CDCA3, and CDK1) peaked at 24-48 h. siRNA-mediated HSP27 silencing in proliferating MRC-5 cells induced G2 arrest coinciding with down-regulation of these six genes. Of note, the promoters of all of these genes have the cell cycle-dependent element and/or the cell cycle gene-homology region. These promoter regions are known to be bound by the E2F family proteins (E2F-1 to E2F-8) and retinoblastoma (RB) family proteins (RB1, p107, and p130), among which E2F-4 and p130 were strongly up-regulated in HSP27-knockdown cells. E2F-4 or p130 knockdown concomitant with the HSP27 knockdown rescued MRC-5 cells from G2 arrest and up-regulated the six cell cycle genes. Moreover, we observed cellular senescence in MRC-5 cells on day 3 after the HSP27 knockdown, as evidenced by increased senescence-associated ß-gal activity and up-regulated inflammatory cytokines. The cellular senescence was also suppressed by the concomitant knockdown of E2F-4/HSP27 or p130/HSP27. Our findings indicate that HSP27 promotes cell cycle progression of MRC-5 cells by suppressing expression of the transcriptional repressors E2F-4 and p130.

Sweet DREAMs for Hippo.

The Hippo pathway coordinates organ size and cell proliferation. The retinoblastoma family of proteins regulates progression through the G0/G1 phase of the cell cycle. Disruption of either pathway contributes to cancer formation. Three recent studies in Genes & Development reveal how cellular proliferation is coordinated between these pathways. Here we discuss the implications of these studies and the new questions that they raise.

DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.

In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence.

Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins.

The human cytomegalovirus (HCMV)-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates the retinoblastoma (Rb) tumor suppressor. Here, we identify the other Rb family members p107 and p130 as novel targets of UL97. UL97 phosphorylates p107 and p130 thereby inhibiting their ability to repress the E2F-responsive E2F1 promoter. As with Rb, this phosphorylation, and the rescue of E2F-responsive transcription, is dependent on the L1 LXCXE motif in UL97 and its interacting clefts on p107 and p130. Interestingly, UL97 does not induce the disruption of all p107-E2F or p130-E2F complexes, as it does to Rb-E2F complexes. UL97 strongly interacts with p107 but not Rb or p130. Thus the inhibitory mechanisms of UL97 for Rb family protein-mediated repression of E2F-responsive transcription appear to differ for each of the Rb family proteins. The immediate early 1 (IE1) protein of HCMV also rescues p107- and p130-mediated repression of E2F-responsive gene expression, but it does not induce their phosphorylation and does not disrupt p107-E2F or p130-E2F complexes. The unique regulation of Rb family proteins by HCMV UL97 and IE1 attests to the importance of modulating Rb family protein function in HCMV-infected cells.

Effect of sex steroid hormone fluctuations in the pathophysiology of male-retinal pigment epithelial cells.

Gender-based differences may influence the occurrence of several ocular conditions suggesting the possibility that fluctuations in sex steroid homeostasis may have direct effects on the eye physiology. Here, we evaluated the effect of sex steroid hormone fluctuations in male retinal pigment epithelial cells, RPEs (ARPE-19). To mimic hormonal fluctuations occurring during aging, we exposed ARPE-19 to acute, prolonged or chronic estradiol, and progesterone challenges. We found that chronic estradiol treatment promotes a remarkable necrosis of RPE cells, and does not affect pRb2/p130 or PAI-2 sub-cellular localization. In contrast, chronic progesterone exposure induces nuclear subcellular rearrangement of pRb2/p130, co-immunolocalization of pRb2/p130 with PAI-2, and accumulation of cells in G2/M phase, which is accompanied by a remarkable reduction of necrosis in favour of apoptosis activation. This study has a high clinical significance since it considers sex steroid fluctuations as inducers of milieu change in the retina able to influence pathological situations occurring with aging in non-reproductive systems such as the eye. Exogenous administration of physiologically significant amounts of sex hormones for long periods of time is a common clinical practice for transgender patients seeking sex reassignment. In particular, our study offers the unique opportunity to unravel the effects of sex hormones, not only in determining gender differences but also in affecting the physiology of non-reproductive systems, such as the eye, in the underserved transgender community.

Nucleosomes positioning around transcriptional start site of tumor suppressor (Rbl2/p130) gene in breast cancer.

Dynamic positioning of nucleosomes is pivotal in determining level of genes expression especially on or around transcription start site (TSS) of a gene. Purpose of the current study was to determine nucleosome position around TSS of Rbl2/p130. We investigated Rbl2/p130 expression in connection to nucleosome positions around its TSS among breast tumors and their adjacent normal control tissues (ANCT) using micrococcal nuclease (MNAse) digestion assay and ChIP-PCR analysis. Three fold reduced Rbl2/p130 expression in these tumor tissues were noticed compared to their control tissues. DNA obtained from MNAse digested chromatin was used as PCR template. Region between - 137 to + 140 around TSS was scanned using 3 primer pairs (P1 = - 137 to + 69; P2 = - 90 to + 69; P3 = - 33 to + 140). ~ 66% breast tumors and ~ 26% ANCT samples were positive for P1. The difference was found statistically significant (p = 0.000) with an odd ratio (OD) of 9.143, suggesting that nucleosome formation in this region is ~ 9 times more probable in tumor samples. ~ 73% of the tumor and 60% ANCT were positive for P2, which although is significant (p = 0.035) with OD = 3.250, but less preferable than P1. However, P3 was not found to be a preferred area for nucleosome occupancy (p = 0.670; OD = 1.2). Negative correlations for nucleosome positions were observed especially for P1. Our results indicate that nucleosome are present slightly downstream of TSS in routine, while in case of breast carcinogenesis nucleosomes slides 55 bases upstream of the TSS, aligning + 1 position at the center of nucleosome, hence hindering access to the transcriptional machinery.

Loss of Rb proteins causes genomic instability in the absence of mitogenic signaling.

Loss of G1/S control is a hallmark of cancer, and is often caused by inactivation of the retinoblastoma pathway. However, mouse embryonic fibroblasts lacking the retinoblastoma genes RB1, p107, and p130 (TKO MEFs) are still subject to cell cycle control: Upon mitogen deprivation, they enter and complete S phase, but then firmly arrest in G2. We now show that G2-arrested TKO MEFs have accumulated DNA damage. Upon mitogen readdition, cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the descendent cell population. Thus, our results demonstrate that unfavorable growth conditions can cause genomic instability in cells lacking G1/S control. This mechanism may allow premalignant tumor cells to acquire additional genetic alterations that promote tumorigenesis.

Genomic landscape of retinoblastoma in Rb-/- p130-/- mice resembles human retinoblastoma.

Several murine retinoblastoma models have been generated by deleting the genes encoding for retinoblastoma susceptibility protein pRb and one of its family members p107 or p130. In Rb-/- p107-/- retinoblastomas, somatic copy number alterations (SCNAs) like Mdm2 amplification or Cdkn2a deletion targeting the p53-pathway occur, which is uncommon for human retinoblastoma. In our study, we determined SCNAs in retinoblastomas developing in Rb-/- p130-/- mice and compared this to murine Rb-/- p107-/- tumors and human tumors. Chimeric mice were made by injection of 129/Ola-derived Rb-/- p130-/- embryonic stem cells into wild type C57BL/6 blastocysts. SCNAs of retinoblastoma samples were determined by low-coverage (∼0.5×) whole genome sequencing. In Rb-/- p130-/- tumors, SCNAs included gain of chromosomes 1 (3/23 tumors), 8 (1/23 tumors), 10 (1/23 tumors), 11 (2/23 tumors), and 12 (4/23 tumors), which could be mapped to frequently altered chromosomes in human retinoblastomas. While the altered chromosomes in Rb-/- p130-/- tumors were similar to those in Rb-/- p107-/- tumors, the alteration frequencies were much lower in Rb-/- p130-/- tumors. Most of the Rb-/- p130-/- tumors (16/23 tumors, 70%) were devoid of SCNAs, in strong contrast to Rb-/- p107-/- tumors, which were never (0/15 tumors) SCNA-devoid. Similarly, to human retinoblastoma, increased age at diagnosis significantly correlated with increased SCNA frequencies. Additionally, focal loss of Cdh11 was observed in one Rb-/- p130-/- tumor, which enforces studies in human retinoblastoma that identified CDH11 as a retinoblastoma suppressor. Moreover, based on a comparison of genes altered in human and murine retinoblastoma, we suggest exploring the role of HMGA1 and SRSF3 in retinoblastoma development. © 2016 Wiley Periodicals, Inc.

The Retinoblastoma (RB) Tumor Suppressor: Pushing Back against Genome Instability on Multiple Fronts.

The retinoblastoma (RB) tumor suppressor is known as a master regulator of the cell cycle. RB is mutated or functionally inactivated in the majority of human cancers. This transcriptional regulator exerts its function in cell cycle control through its interaction with the E2F family of transcription factors and with chromatin remodelers and modifiers that contribute to the repression of genes important for cell cycle progression. Over the years, studies have shown that RB participates in multiple processes in addition to cell cycle control. Indeed, RB is known to interact with over 200 different proteins and likely exists in multiple complexes. RB, in some cases, acts through its interaction with E2F1, other members of the pocket protein family (p107 and p130), and/or chromatin remodelers and modifiers. RB is a tumor suppressor with important chromatin regulatory functions that affect genomic stability. These functions include the role of RB in DNA repair, telomere maintenance, chromosome condensation and cohesion, and silencing of repetitive regions. In this review we will discuss recent advances in RB biology related to RB, partner proteins, and their non-transcriptional functions fighting back against genomic instability.

Cortical excitatory neurons become protected from cell division during neurogenesis in an Rb family-dependent manner.

Cell cycle dysregulation leads to abnormal proliferation and cell death in a context-specific manner. Cell cycle progression driven via the Rb pathway forces neurons to undergo S-phase, resulting in cell death associated with the progression of neuronal degeneration. Nevertheless, some Rb- and Rb family (Rb, p107 and p130)-deficient differentiating neurons can proliferate and form tumors. Here, we found in mouse that differentiating cerebral cortical excitatory neurons underwent S-phase progression but not cell division after acute Rb family inactivation in differentiating neurons. However, the differentiating neurons underwent cell division and proliferated when Rb family members were inactivated in cortical progenitors. Differentiating neurons generated from Rb(-/-); p107(-/-); p130(-/-) (Rb-TKO) progenitors, but not acutely inactivated Rb-TKO differentiating neurons, activated the DNA double-strand break (DSB) repair pathway without increasing trimethylation at lysine 20 of histone H4 (H4K20), which has a role in protection against DNA damage. The activation of the DSB repair pathway was essential for the cell division of Rb-TKO differentiating neurons. These results suggest that newly born cortical neurons from progenitors become epigenetically protected from DNA damage and cell division in an Rb family-dependent manner.

HPV 16E7 and 48E7 proteins use different mechanisms to target p130 to overcome cell cycle block.

BACKGROUND: Retinoblastoma like protein 2 (RBL2) or p130 is a member of the pocket protein family, which is infrequently mutated in human tumours. Its expression is posttranscriptionally regulated and largely G0 restricted. We have previously shown that E6/E7 oncoproteins encoded by human papillomavirus (HPV) type 16, which is a high-risk type for cervical cancer development, must target p130 to promote the host cell to exit from quiescence (G0) state and enter S phase of the cell cycle. P130 is associated with the DREAM (DP, RB-like, E2F and MuvB) complex in G0/G1, which prevents S phase progression by repressing transcription of E2F-regulated genes. E7 proteins could potentially disrupt the p130-DREAM complex through two known mechanisms: direct interaction with p130 or induction of cyclin dependent kinase 2 (CDK2) phosphorylation by interacting with its inhibitor, p21(CIP1). METHODS: In this study we have used p130 mutants deficient in binding the E7 LXCXE domain (p130mE7), unphosphorylatable by CDK2 (p130PM22) or a combination of both (p130PM22/mE7) to investigate these mechanisms used by E7 proteins to disrupt the p130-DREAM complex and promote cell cycle progression. RESULTS: We found that HPV16 E7 binding to p130 through its LXCXE domain was absolutely required to disrupt p130-DREAM to promote S phase of the cell cycle, as HPV16 E7 was unable to suppress p130mE7 but could suppress p130PM22. In contrast, the E7 protein encoded by a cutaneous HPV type that lacks a functional LXCXE domain, HPV 48 E7, was also able to disrupt p130-DREAM to promote cell cycling, but through the alternative mechanism. Thus, HPV48 E7 could suppress a cell cycle block imposed by p130mE7, but was unable to suppress p130PM22. CONCLUSIONS: Overall, these results indicate that suppression of p130 is required for HPV-induced cell cycling, and that different HPV E7 proteins can use alternative mechanisms to achieve this.