P18Ink4c and p53 Act as tumor suppressors in cyclin D1-driven primitive neuroectodermal tumor.

miR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Mutations in the Retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1,2,3. Despite being the first tumour suppressor to be identified, the molecular and cellular basis underlying selection for persistent RB loss in cancer remains unclear4–6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and currently under evaluation in lung adenocarcinoma7–9. Whether RB pathway reactivation will have therapeutic effects and if targeting CDK4/6 is sufficient to reactivate RB pathway activity in lung cancer is unknown. Here, we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in the mouse. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for MAPK signal amplification during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to CDK4/6 inhibition. Second, RB inactivation deregulates expression of cell state-determining factors, facilitates lineage infidelity, and accelerates the acquisition of metastatic competency. In contrast, reactivation of RB reprograms advanced tumours toward a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs they control, and critical determinants of successful therapy.

he retinoblastoma (RB) tumor suppressor is believed to play an important role in the pathogenesis of osteosarcomas. Loss of function of RB may occur through abrogation of the RB/p16/cyclin Dl regularity pathway. Our study evaluated the protein expression of pRB, p16, and cyclin D1 in osteosarcomas: 27 tumor specimens from 20 patients were immunostained with antibodies to pRB, p16, and cyclin Dl using the avidin-biotin method with antigen retrieval. In a subset of cases, the immuno-histochemical expression of pRB was correlated with loss of heterozygosity (LOH) at the RB locus; 56% of the specimens (15/27 cases) had an altered component of the pRB Ip16/cyclin D1 pathway with pRB the most frequent abnormal protein (9/27 or 33%) and p16 and cyclin D1 immunohistochemically abnormal in 2 and 3 patients, respectively. Only one protein in the pathway was abnormally expressed in any individual specimen. Absent pRB expression was associated with LOH in 7 of 15 cases (47%), white in another 8 cases pRB was expressed immunohistochemically despite LOH noted by molecular analysis. Two cases were heterozygous by molecular analysis and demonstrated positive nuclear staining. These results support the role of the RB cell cycle control pathway (pRB, p16, cyclin D1) in the pathogenesis of a subset of osteosarcomas. The results also seem to identify a subset of osteosarcomas (about one-third in our study) in which other genetic factors, including others in this pathway, may be important.

The p53 family includes three members that share significant sequence homology, yet exhibit fundamentally different functions in tumorigenesis. Whereas p53 displays all characteristics of a classical tumor suppressor, its homologues p63 and p73 do not. We have previously shown, that NH(2)-terminally truncated isoforms of p73 (Delta TA-p73), which act as dominant-negative inhibitors of p53 are frequently overexpressed in cancer cells. Here we provide evidence that Delta TA-p73 isoforms also affect the retinoblastoma protein (RB) tumor suppressor pathway independent of p53. Delta TA-p73 isoforms inactivate RB by increased phosphorylation, resulting in enhanced E2F activity and proliferation of fibroblasts. By inactivating the two major tumor suppressor pathways in human cells they act functionally analogous to several viral oncoproteins. These findings provide an explanation for the fundamentally different functions of p53 and p73 in tumorigenesis.

Medulloblastoma, which accounts for 20-25% of all childhood brain tumors, is defined as a primitive neuroectodermal tumor (PNET) located in the cerebellum. Supratentorial PNET are less frequent than medulloblastoma. But their clinical outcome is worse than in medulloblastomas. Chromosome 10q contains at least 2 tumor suppressor genes that might play a role in brain tumor development: PTEN and DMBT1. The aim of this study was to compare the status of homozygous deletion and expression of PTEN and DMBT1 genes in PNET primary tumor samples and cell lines. Homozygous deletions of PTEN and DMBT1 were studied in 32 paraffin-embedded PNET samples (23 medulloblastomas and 9 supratentorial PNET) and in 7 PNET cell lines, by differential PCR and by FISH. PTEN homozygous losses were demonstrated in 7 medulloblastomas (32%) and in no supratentorial PNET, while homozygous deletions of DMBT1 appeared in 1 supratentorial PNET (20%) and in 7 medulloblastomas (33%). No homozygous deletion of PTEN or DMBT1 was detected in any of the PNET cell lines either by differential PCR or by FISH. Expression study of the 2 genes was performed in the 7 PNET cell lines by RT-PCR. One PNET cell line lacked PTEN and DMBT1 expression, while 2 medulloblastoma cell lines did not express DMBT1. Our results add some positive data to the hypothesis that supratentorial PNETs and medulloblastomas might be genetically different.

The recently described multiple tumor suppressor 1/cyclin-dependent kinase inhibitor 2 (MTS1/CDKN2) gene, encoding the cyclin-dependent kinase 4 inhibitor p16, is mutated in a wide variety of tumor cell lines, including gliomas. To investigate the possible role of this gene in the genesis of the central nervous system primitive neuroectodermal tumor (PNET), four established PNET cell lines and 18 PNET surgical specimens were studied for deletions and mutations of the MTS1/CDKN2 gene. One of the four cell lines had homozygous deletion of the gene. No mutation in any of the three MTS1/CDKN2 exons was detected in the other three cell lines by single strand conformational polymorphism analysis. Eighteen surgical PNET specimens were studied for allelic and homozygous deletion at chromosome 9p21, the location of the MTS1/CDKN2 gene. No loss of heterozygosity was noted in 11 of the tumors, and no homozygous loss was noted in any tumor. Single strand conformational polymorphism analysis of the entire coding region of the MTS1/CDKN2 gene revealed no mutation within MTS1/CDKN2 in any tumor. Although deletion of MTS1/CDKN2 may occur in some PNET cell lines, neither deletion nor mutation of the gene is found in tumors before culture. The genesis of the human central nervous system PNET does not involve deletion or mutation of the MTS1/CDKN2 gene.

A New Pineoblastoma Cell Line, PER-480, with der(10)t(10;17), der(16)t(1;16), and Enhanced MYC Expression in the Absence of Gene Amplification

Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription.

Zhong et al., pp. 2125–2131 It is thought that only a minority of cells within any tumor have the ability to self-renew. In the context of retinoblastoma (RB), the consensus view is that RB cells originate from retinal stem cells (RSC) or multipotent primitive retinoblasts. There is, however, no direct evidence that tumorigenic RSCs actually exist within RBs. Previously, Zhong et al. had successfully cultivated stem-like cells from human RBs. In this report they have taken the results a step further by investigating whether these retinal stem-like cells (RSLCs) have the potential to reproduce the parental tumor in vivo. Using tumor tissues from eight patients with either differentiated or nondifferentiated RB, primary cell cultures were generated under conditions that favored stem cell growth. All tumor-derived cultures developed nonadherent neurospheres that could be passaged many times. These cells were found to be capable of self-renewal and expressed neural stem cell markers such as pax6, nestin and Bmi-1. RSLCs also exhibited an impressive degree of multipotentiality, forming neurons and glial cells – among others – though they fell short of the full cellular repertoire of RSCs. After intraocular (I.O.) injection of four of the RB primary cultures into NOD/SCID mice, the mice developed tumors within 2–12 weeks. Interestingly, these xenograft tumors were very similar to their parental tumor cells. To further confirm that these xenografts contain RSLCs, single tumor cells were isolated from the xenograft tumors and reinjected I.O. into new animals, which in turn developed RB-like tumors. These findings strongly suggest that RB derives from RSLCs. In addition to furthering our understanding of RB tumorigenesis, this model could be useful in developing new therapeutic approaches for the disease. Vickers et al., pp. 2212–2217 Total prostate specific antigen (tPSA) levels can be raised in benign prostate disease, particularly in older patients, complicating its usefulness as a predictor of prostate cancer. Previous studies have shown that levels of human kallikrein-2 (hK2) and the ratio of free to total PSA (%fPSA) may also reflect underlying prostate malignancy. In this study, Vickers et al., wanted to determine whether the combination of these biomarkers could offer a better means of detecting prostate cancer and whether the biomarkers vary as a function of age. Using blood samples from a very large (13,600) cohort of men between the ages of 44 and 50 and 59 and 61 and data from the Swedish Cancer Registry, the authors determined odds ratios and the predictive probability of the various biomarkers in over 500 prostate cancer patients. They found that the predictive value of tPSA is inversely associated with age but not with time of diagnosis. The predictive value of %fPSA combined with hK2 was inversely related to the time of diagnosis but not modified by age. Using tPSA, %fPSA and hK2 compared to tPSA alone enhanced prediction levels in older patients but not younger ones. The results from this study importantly show the value of screening younger men (<50) for PSA and that combined biomarker testing may be needed to detect prostate malignancy more accurately in older subjects. Inskip et al., pp. 2233–2240 Mortalities associated with childhood cancers have decreased over the last few decades. However, the aggressive nature of the treatments means that patients are subsequently at risk of developing new primary cancers. Setting up a large-scale follow-up of childhood cancer survivors is not an easy task. In this report, Inskip et al. use a US population-based cancer registry — SEER – which contains data on nearly 26,000 childhood cancer survivors from 1973–2002, to determine new malignancy risk in childhood cancer patients. Using observed to expected ratios and regression analysis, the cancer survivors were found to have an almost 6-fold increased risk of developing a new primary cancer compared to the general population. The degree of risk and nature of the second cancer was highly dependent on the first type of cancer the patient had suffered from. Hodgkin lymphoma (HL), retinoblastoma, primitive neuroectodermal tumor and Ewing sarcoma were the cancers associated with the highest risk of developing a second primary cancer. The most likely subsequent cancers were female breast, CNS, bone, thyroid, soft tissue, melanoma and acute nonlymphocytic leukemia (ANLL). The mode of original cancer treatment also had an influence on subsequent cancer development. Patients who had received radiotherapy were more likely to develop a solid tumor, whereas patients treated with chemotherapy were at higher risk of developing ANLL. This thorough study gives a comprehensive analysis of the new malignancy risks facing survivors of childhood cancer. This perspective and future follow-up studies will be important to help monitor patients and evaluate the risks associated with changing cancer treatments.

The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.

Crosstalk Between Cyclins D1 and D3 in Mantle Cell Level At the Transcriptional and Postranscriptional Levels

p53 is a well-established critical cell cycle regulator. By inducing transcription of the gene encoding p21, p53 inhibits cyclin-dependent kinase (CDK)-mediated phosphorylation of cell cycle inhibitor retinoblastoma (RB) proteins. Phosphorylation of RB releases E2F transcription factor proteins that transactivate cell cycle-promoting genes. Here, we sought to uncover the contribution of p53, p21, CDK, RB, and E2F to the regulation of ferroptosis, an oxidative form of cell death. Our studies have uncovered unexpected complexity in this regulation. First, we showed that elevated levels of p53 enhance ferroptosis in multiple inducible and isogenic systems. On the other hand, we found that p21 suppresses ferroptosis. Elevation of CDK activity also suppressed ferroptosis under conditions where p21 suppressed ferroptosis, suggesting that the impact of p21 must extend beyond CDK inhibition. Furthermore, we showed that overexpression of E2F suppresses ferroptosis in part via a p21-dependent mechanism, consistent with reports that this transcription factor can induce transcription of p21. Finally, deletion of RB genes enhanced ferroptosis. Taken together, these results show that signals affecting ferroptotic sensitivity emanate from multiple points within the p53 tumor suppressor pathway.

Deregulation of the cell division cycle is critical for cancer development. Transition from G1 to S phase of the cell cycle is promoted by D-type cyclins (D1, D2 and D3). Cyclin D1 is a critical regulator of cell cycle progression due to its ability to activate cyclin dependent kinases (CDK) 4 and 6, which phosphorylate and inactivate the retinoblastoma protein (pRb) and activates the transcription factor E2F1 to promote the expression of genes essential for G1/S progression. Importantly, mutations in the tumor suppressor gene RB1 or inactivation of the RB tumor suppressor pathway occurs in almost all human malignancies and one such mechanism involves cyclin D1 overexpression. Even in cancer cells with amplified or elevated cyclin D1 mRNA, the Cyclin D1 protein rapidly declines as cells progress through the G1 phase of the cell cycle to reach undetectable levels in S-phase, and this is thought to be dependent on the Skp1-Cul1-F-box protein (SCF) E3 ubiquitin ligase complex. The identity of the E3 ubiquitin ligase that is specifically involved in downregulating cyclin D1 during S-phase of the cell cycle or the functional significance of cyclin D1 downregulation however, remains unknown, as recent genetic studies in mice demonstrated that the steady state levels or stability of cyclin D1 protein, are not altered following the inactivation of the known regulators of cyclin D1 stability. Using a genetic si-RNA-based screen, we have identified a novel E3 ubiquitin ligase; SCF-FBXW5, which specifically interacts with and downregulates cyclin D1 protein, both in unperturbed proliferating cancer cells and following DNA damage. Importantly, inactivation of this E3 ubiquitin ligase inhibits the proliferation of cancer cells primarily through cyclin D1-dependent inhibition of S-phase progression and sensitizes cancer cells to DNA damage induced by UV or ionizing radiation. These findings identify a novel axis of regulating cyclin D1 stability in cancer cells with implications for targeting this axis to inhibit cyclin D1-driven cancers. Citation Format: Fadila Guessous, Jinho Heo, Naga Vaddadi, Tarek Abbas. Novel regulation of cyclin D1 stability and the DNA damage response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2015-3786

Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

The primitive neuroectodermal tumor of the central nervous system is one of a number of tumors in which deletions on chromosome 17p have been identified. The tumor suppressor gene, p53, is located in the region of the deletion. To determine if the p53 gene is involved in the development of primitive neuroectodermal tumors, deoxyribonucleic acid (DNA) blot analysis, ribonucleic acid blot analysis, and p53 complementary DNA sequencing were performed on 34 primitive neuroectodermal tumors removed from children. No rearrangement in the gene was detected in 21 tumors. The p53 messenger ribonucleic acid was of the expected size in all 18 tumors for which ribonucleic acid was available. Sequencing of p53 Exons 5 through 9 revealed a mutation in the cell line DAOY and in only 1 of 14 tumors examined. A DNA rearrangement was detected in the DNA from one tumor with a probe mapping to the distal portion of 17p. Taken together, these data suggest that the p53 gene is not involved in the development of most primitive neuroectodermal tumors. In addition, a gene of interest may be present on distal 17p.