Sanja Pajovic

Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies.

BACKGROUND Retinoblastoma is the childhood retinal cancer that defined tumour-suppressor genes. Previous work shows that mutation of both alleles of the RB1 retinoblastoma suppressor gene initiates disease. We aimed to characterise non-familial retinoblastoma tumours with no detectable RB1 mutations. METHODS Of 1068 unilateral non-familial retinoblastoma tumours, we compared those with no evidence of RB1 mutations (RB1(+/+)) with tumours carrying a mutation in both alleles (RB1(-/-)). We analysed genomic copy number, RB1 gene expression and protein function, retinal gene expression, histological features, and clinical data. FINDINGS No RB1 mutations (RB1(+/+)) were reported in 29 (2·7%) of 1068 unilateral retinoblastoma tumours. 15 of the 29 RB1(+/+) tumours had high-level MYCN oncogene amplification (28-121 copies; RB1(+/+)MYCN(A)), whereas none of 93 RB1(-/-) primary tumours tested showed MYCN amplification (p<0·0001). RB1(+/+)MYCN(A) tumours expressed functional RB1 protein, had fewer overall genomic copy-number changes in genes characteristic of retinoblastoma than did RB1(-/-) tumours, and showed distinct aggressive histological features. MYCN amplification was the sole copy-number change in one RB1(+/+)MYCN(A) retinoblastoma. One additional MYCN(A) tumour was discovered after the initial frequencies were determined, and this is included in further analyses. Median age at diagnosis of the 17 children with RB1(+/+)MYCN(A) tumours was 4·5 months (IQR 3·5-10), compared with 24 months (15-37) for 79 children with non-familial unilateral RB1(-/-) retinoblastoma. INTERPRETATION Amplification of the MYCN oncogene might initiate retinoblastoma in the presence of non-mutated RB1 genes. These unilateral RB1(+/+)MYCN(A) retinoblastomas are characterised by distinct histological features, only a few of the genomic copy-number changes that are characteristic of retinoblastoma, and very early age of diagnosis. FUNDING National Cancer Institute-National Institutes of Health, Canadian Institutes of Health Research, German Research Foundation, Canadian Retinoblastoma Society, Hyland Foundation, Toronto Netralaya and Doctors Lions Clubs, Ontario Ministry of Health and Long Term Care, UK-Essen, and Foundations Avanti-STR and KiKa.

Kinesin family member 14: An independent prognostic marker and potential therapeutic target for ovarian cancer

The novel oncogene KIF14 (kinesin family member 14) shows genomic gain and overexpression in many cancers including OvCa (ovarian cancer). We discovered that expression of the mitotic kinesin KIF14 is predictive of poor outcome in breast and lung cancers. We now determine the prognostic significance of KIF14 expression in primary OvCa tumors, and evaluate KIF14 action on OvCa cell tumorigenicity in vitro. Gene‐specific multiplex PCR and real‐time QPCR were used to measure KIF14 genomic (109 samples) and mRNA levels (122 samples) in OvCa tumors. Association of KIF14 with clinical variables was studied using Kaplan–Meier survival and Cox regression analyses. Cellular effects of KIF14 overexpression (stable transfection) and inhibition (stable shRNA knockdown) were studied by proliferation (cell counts), survival (Annexin V immunocytochemistry) and colony formation (soft‐agar growth). KIF14 genomic gain (>2.6 copies) was present in 30% of serous OvCas, and KIF14 mRNA was elevated in 91% of tumors versus normal epithelium. High KIF14 in tumors independently predicted for worse outcome (p = 0.03) with loss of correlation with proliferation marker expression and increased rates of recurrence. Overexpression of KIF14 in OvCa cell lines increased proliferation and colony formation (p < 0.01), whereas KIF14 knockdown induced apoptosis and dramatically reduced colony formation (p < 0.05). Our findings indicate that KIF14 mRNA is an independent prognostic marker in serous OvCa. Dependence of OvCa cells on KIF14 for maintenance of in vitro colony formation suggests a role of KIF14 in promoting a tumorigenic phenotype, beyond its known role in proliferation.

Cdh11 Acts as a Tumor Suppressor in a Murine Retinoblastoma Model by Facilitating Tumor Cell Death

CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01). Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8) (p = 0.01) and had fewer multifocal tumors at PND28 (p = 0.016), compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003). In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p<0.03 for all markers), while proliferation in vivo remained unaffected (p = 0.121). Activated caspase-3 was significantly decreased and β-catenin expression increased in Cdh11 knockdown experiments in vitro. These data suggest that Cdh11 displays tumor suppressor properties in vivo and in vitro in murine retinoblastoma through promotion of cell death.

Loss of p75 neurotrophin receptor expression accompanies malignant progression to human and murine retinoblastoma.

We studied the expression of pro‐apoptotic neurotrophin receptor p75 (p75NTR) in human and murine retinoblastoma, compared to normal retina, and examined changes in p75NTR expression with the onset of apoptosis in the course of murine retinoblastoma progression, using immunohistochemistry and quantitative real‐time RT‐PCR. The murine retinoblastoma is induced by retinal specific expression of SV40 T‐antigen (TAg), which blocks the function of the retinoblastoma protein (pRB) and related proteins, and is a well‐studied model that closely simulates human retinoblastoma. The majority of human retinoblastoma either lacked or expressed decreased levels of p75NTR mRNA, compared to human retina. Moreover, p75NTR protein was not detected in any tumor studied, unlike normal retina. Like human retinoblastoma, advanced murine retinoblastoma did not express p75NTR. However, before tumors emerged, small clusters of TAg‐positive cells coexpressed p75NTR and activated caspase‐3, a marker of apoptosis. Furthermore, in three rare human eyes containing retinoblastoma adjacent to regions resembling the benign retinal tumor retinoma, both normal retina and retinoma‐like tissue expressed p75NTR protein, while the retinoblastoma did not. We suggest that p75NTR loss accompanies progression from retinoma to retinoblastoma.

Regulation of p14ARF expression by miR-24: a potential mechanism compromising the p53 response during retinoblastoma development

BackgroundMost human cancers show inactivation of both pRB- and p53-pathways. While retinoblastomas are initiated by loss of the RB1 tumor suppressor gene, TP53 mutations have not been found. High expression of the p53-antagonist MDM2 in human retinoblastomas may compromise p53 tumor surveillance so that TP53 mutations are not selected for in retinoblastoma tumorigenesis. We previously showed that p14ARF protein, which activates p53 by inhibiting MDM2, is low in retinoblastomas despite high mRNA expression.MethodsIn human fetal retinas, adult retinas, and retinoblastoma cells, we determined endogenous p14ARF mRNA, ARF protein, and miR-24 expression, while integrity of p53 signalling in WERI-Rb1 cells was tested using an adenovirus vector expressing p14ARF. To study p14ARF biogenesis, retinoblastoma cells were treated with the proteasome inhibitor, MG132, and siRNA against miR-24.ResultsIn human retinoblastoma cell lines, p14ARF mRNA was disproportionally high relative to the level of p14ARF protein expression, suggesting a perturbation of p14ARF regulation. When p14ARF was over-expressed by an adenovirus vector, expression of p53 and downstream targets increased and cell growth was inhibited indicating an intact p14ARF-p53 axis. To investigate the discrepancy between p14ARF mRNA and protein in retinoblastoma, we examined p14ARF biogenesis. The proteasome inhibitor, MG132, did not cause p14ARF accumulation, although p14ARF normally is degraded by proteasomes. miR-24, a microRNA that represses p14ARF expression, is expressed in retinoblastoma cell lines and correlates with lower protein expression when compared to other cell lines with high p14ARF mRNA. Transient over-expression of siRNA against miR-24 led to elevated p14ARF protein in retinoblastoma cells.ConclusionsIn retinoblastoma cells where high levels of p14ARF mRNA are not accompanied by high p14ARF protein, we found a correlation between miR-24 expression and low p14ARF protein. p14ARF protein levels were restored without change in mRNA abundance upon miR-24 inhibition suggesting that miR-24 could functionally repress expression, effectively blocking p53 tumor surveillance. During retinal tumorigenesis, miR-24 may intrinsically compromise the p53 response to RB1 loss.

Expression of p14ARF, MDM2, and MDM4 in human retinoblastoma.

It is still not clear whether the p53 pathway is altered in retinoblastoma development. We assessed the expression of the p53 pathway genes p14(ARF), mouse double minute 2 (MDM2), and mouse double minute 4 (MDM4) in human retinoblastoma compared to normal retina. Primary human retinoblastomas, retinoblastoma cell lines and normal retinas were assessed for p14(ARF) and MDM4 mRNA by quantitative RT-PCR. p14(ARF), MDM2, and MDM4 protein were measured by immunoblot and immunohistochemistry. Compared to retina, p14(ARF) mRNA expression was notably increased in retinoblastoma but p14(ARF) protein was undetectable. MDM2 and MDM4 proteins were expressed in 22/22 retinoblastomas. MDM2 was expressed in 3/10 retinas tested, and MDM4 in 10/10 retinas. The expression level of MDM2 protein in retinoblastomas and retina was comparable, while MDM4 protein was overexpressed in one retinoblastoma cell line Y79 and two primary retinoblastomas. We observe that overexpression of MDM2 and MDM4 is not a necessary step in retinoblastoma development. However, loss of detectable p14(ARF) protein and resultant lack of functional inactivation of these p53 inhibitors may contribute to retinoblastoma development by constitutive inhibition of p53.

The TAg-RB Murine Retinoblastoma Cell of Origin Has Immunohistochemical Features of Differentiated Muller Glia with Progenitor Properties

PURPOSE Human retinoblastoma arises from an undefined developing retinal cell after inactivation of RB1. This is emulated in a murine retinoblastoma model by inactivation of pRB by retinal-specific expression of simian virus 40 large T-antigen (TAg-RB). Some mutational events after RB1 loss in humans are recapitulated at the expression level in TAg-RB, supporting preclinical evidence that this model is useful for comparative studies between mouse and human. Here, the characteristics of the TAg-RB cell of origin are defined. METHODS TAg-RB mice were killed at ages from embryonic day (E)18 to postnatal day (P)35. Tumors were analyzed by immunostaining, DNA copy number PCR, or real-time quantitative RT-PCR for TAg protein, retinal cell type markers, and retinoblastoma-relevant genes. RESULTS TAg expression began at P8 in a row of inner nuclear layer cells that increased in number through P21 to P28, when clusters reminiscent of small tumors emerged from cells that escaped a wave of apoptosis. Early TAg-expressing cells coexpressed the developmental marker Chx10 and glial markers CRALBP, clusterin, and carbonic anhydrase II (Car2), but not TuJ1, an early neuronal marker. Emerging tumors retained expression of only Chx10 and carbonic anhydrase II. As with human retinoblastoma, TAg-RB tumors showed decreased Cdh11 DNA copy number and gain of Kif14 and Mycn. It was confirmed that TAg-RB tumors lose expression of tumor suppressor cadherin-11 and overexpress oncogenes Kif14, Dek, and E2f3. CONCLUSIONS TAg-RB tumors displayed molecular similarity to human retinoblastoma and origin in a cell with features of differentiated Müller glia with progenitor properties.

Transcriptional and Epigenetic Regulation of KIF14 Overexpression in Ovarian Cancer

KIF14 (kinesin family member 14) is a mitotic kinesin and an important oncogene in several cancers. Tumor KIF14 expression levels are independently predictive of poor outcome, and in cancer cells KIF14 can modulate metastatic behavior by maintaining appropriate levels of cell adhesion and migration proteins at the cell membrane. Thus KIF14 is an exciting potential therapeutic target. Understanding KIF14s regulation in cancer cells is crucial to the development of effective and selective therapies to block its tumorigenic function(s). We previously determined that close to 30% of serous ovarian cancers (OvCa tumors) exhibit low-level genomic gain, indicating one mechanism of KIF14 overexpression in tumors. We now report on transcriptional and epigenetic regulation of KIF14. Through promoter deletion analyses, we identified one cis-regulatory region containing binding sites for Sp1, HSF1 and YY1. siRNA-mediated knockdown of these transcription factors demonstrated endogenous regulation of KIF14 overexpression by Sp1 and YY1, but not HSF1. ChIP experiments confirmed an enrichment of both Sp1 and YY1 binding to the endogenous KIF14 promoter in OvCa cell lines with high KIF14 expression. A strong correlation was seen in primary serous OvCa tumors between Sp1, YY1 and KIF14 expression, further evidence that these transcription factors are important players in KIF14 overexpression. Hypomethylation patterns were observed in primary serous OvCa tumors, suggesting a minor role for promoter methylation in the control of KIF14 gene expression. miRNA expression analysis determined that miR-93, miR-144 and miR-382 had significantly lower levels of expression in primary serous OvCa tumors than normal tissues; treatment of an OvCa cell line with miRNA mimics and inhibitors specifically modulated KIF14 mRNA levels, pointing to potential novel mechanisms of KIF14 overexpression in primary tumors. Our findings reveal multiple mechanisms of KIF14 upregulation in cancer cells, offering new targets for therapeutic interventions to reduce KIF14 in tumors, aiming at improved prognosis.

Retinoblastoma: the evidence does matter.

We are responding to a letter that appeared in your recent issue [1], which contains a number of major inaccuracies regarding retinoblastoma. Similar speculations and misleading conclusions can be found in two previous papers by the same first author [2,3]. We feel that we can provide enough evidence and expertise from our own work to challenge such misconstructions. We provide genetic testing for mutations in the RB1 gene (MIM 180200) through a not-for-profit corporation, Retinoblastoma Solutions. Patient samples are sent to us from around the world (22 countries, 6 continents) and to date we have served over 1080 unique retinoblastoma families. Our own and our collaborators’ research has contributed to the understanding of the mechanisms of retinoblastoma, and cancer in general. Mastrangelo and colleagues state that “. . . in a large number of bilateral cases, no mutations [of the RB1 gene] can be found [. . .] even with the most advanced molecular techniques” [1] (emphasis ours). We do not agree. In 95% (414/436) of the bilateral cases referred to us, a constitutional RB1 mutation is found. There are known categories of mutations which our methods currently cannot detect. The small proportion of remaining cases is predicted to carry mutations in the RB1 gene such as damaging translocations, deep intronic mutations, or presently undetectable mosaic mutations. The estimated proportion of currently undetected mosaic mutations alone is predicted to account for the missing 5% of mutations in the blood of persons with bilateral retinoblastoma [4]. In addition we have found mutations in both alleles of 93% (381/408) of retinoblastoma tumors from unilaterally affected patients. This very high sensitivity to find the RB1 mutation in each family has provided key information for families that not only assists with early (including prenatal) diagnosis resulting in vastly improved vision outcomes, but also decreases the overall cost of health care and improves quality of life by reducing the intensity of surveillance examinations for child relatives of the proband who are proven to not carry the proband’s RB1 mutation. In a recent article [2], Mastrangelo and colleagues state that “[. . .] the mutation rate within the newly diagnosed cases of retinoblastoma, is more likely to approach the 50% more recently reported by Nichols et al.”. This represents a distortion of the results presented in the referenced article. Nichols et al. [5] found RB1 mutations in blood samples from 91% (77/85) of patients with bilateral retinoblastoma, 70% (7/10) of familial unilateral patients and 7% (6/85) of patients with sporadic unilateral retinoblastoma. The 50% sensitivity quoted by Mastrangelo et al. is only achieved by ignoring the accepted biological differences between the three categories and treating them as one homogeneous set. Mastrangelo and colleagues state that their “personal series of retinoblastoma survivors (unpublished data), clearly show [. . .] 50% affected children in the offspring of unilateral retinoblastoma survivors”. This is a very unusual series of unilateral patients. It has long been noted that between 2–6% of all offspring of patients with sporadic unilateral retinoblastoma will develop retinoblastoma [6–8]. Our data affirm the 6% risk, since 13% (53/408) of unilaterally affected patients carry in their blood one of the RB1 mutations found in the tumor, putting them into the hereditary category with up to 50% risk to each of their offspring to develop retinoblastoma. Of the 87% of unilaterally affected individuals shown to not carry either of the tumor RB1 mutations in blood, none have reported a child with retinoblastoma. We would be pleased to examine (for RB1 mutations) the blood of the unilateral patients and their offspring with retinoblastoma that Dr. Mastrangelo describes. We predict some will be heterozygous for RB1 mutations and almost 30% of these will be mosaic for an RB1 mutation that will be heterozygous in their offspring, consistent with Knudson’s two-hit model. We have shown that loss of both alleles of RB1 initiates non-proliferative retinomas (benign retinal tumors), a predicted precursor to retinoblastoma [9–11].

Abstract A18: Uncovering molecular subgroups and a novel cancer driver, ACVR1, in diffuse intrinsic pontine gliomas

Diffuse intrinsic pontine glioma (DIPG) is a devastating paediatric brain tumor with no effective therapy and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and choosing therapies based on assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unravelled the unique genetic make-up of this brain cancer with nearly 80% harbouring a K27M-H3.3 or K27M-H3.1 mutation. However, DIPGs are still thought of as one disease with limited understanding of the genetic drivers of these tumors. Here we apply methylation profiling, whole genome sequencing, expression profiling, and copy number analysis to discover that DIPGs are three molecularly distinct subgroups (H3-K27M, Silent, and MYCN) and uncover a mutations in a novel driver, ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of downstream activin signalling targets ID1 and ID2. The MYCN subgroup is not associated with histone mutations and is instead characterized by hypermethylation and chromothripsis of chromosome 2p with high-level amplifications of MYCN, ID2, and KIDINS220. The Silent subgroup affects younger children, has genomes with minimal genomic instability and fewer mutations, over-expresses WNT pathway genes, as well as genes with known cancer association such as MDM2, MSMP and ADAM33. The H3-K27M subgroup is highly K27M-H3 mutated and associated with additional hits including activating mutations in ACVR1, frequent RB1 and TP53 deletions, PVT-1/MYC or PDGFRA gains/amplifications, genomic instability and alternative lengthening of telomeres. Our results show that this seemingly homogeneous entity in fact comprises three distinct subgroups with different demographic and molecular features. This complexity needs to be considered when designing new therapeutic approaches in order to improve outcome for these children. Citation Format: Pawel Buczkowicz, Christine Hoeman, Patricia Rakopoulos, Sanja Pajovic, Andrew Morrison, Eric Bouffet, Ute Bartels, Oren Becher, Cynthia Hawkins. Uncovering molecular subgroups and a novel cancer driver, ACVR1, in diffuse intrinsic pontine gliomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A18.