Retinoblastoma has properties of a cone precursor tumor and depends upon cone-specific MDM2 signaling.

Retinoblastomas result from the inactivation of the RB1 gene and the loss of Rb protein, yet the cell type in which Rb suppresses retinoblastoma and the circuitry that underlies the need for Rb are undefined. Here, we show that retinoblastoma cells express markers of postmitotic cone precursors but not markers of other retinal cell types. We also demonstrate that human cone precursors prominently express MDM2 and N-Myc, that retinoblastoma cells require both of these proteins for proliferation and survival, and that MDM2 is needed to suppress ARF-induced apoptosis in cultured retinoblastoma cells. Interestingly, retinoblastoma cell MDM2 expression was regulated by the cone-specific RXRgamma transcription factor and a human-specific RXRgamma consensus binding site, and proliferation required RXRgamma, as well as the cone-specific thyroid hormone receptor-beta2. These findings provide support for a cone precursor origin of retinoblastoma and suggest that human cone-specific signaling circuitry sensitizes to the oncogenic effects of RB1 mutations.

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.

Crizotinib in ALK-rearranged inflammatory myofibroblastic tumor.

Inflammatory myofibroblastic tumor (IMT) is a distinctive mesenchymal neoplasm characterized by a spindle-cell proliferation with an inflammatory infiltrate. Approximately half of IMTs carry rearrangements of the anaplastic lymphoma kinase (ALK) locus on chromosome 2p23, causing aberrant ALK expression. We report a sustained partial response to the ALK inhibitor crizotinib (PF-02341066, Pfizer) in a patient with ALK-translocated IMT, as compared with no observed activity in another patient without the ALK translocation. These results support the dependence of ALK-rearranged tumors on ALK-mediated signaling and suggest a therapeutic strategy for genomically identified patients with the aggressive form of this soft-tissue tumor. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Targeted pharmacologic inhibition of S-phase kinase-associated protein 2 (SKP2) mediated cell cycle regulation in lung and other RB-Related cancers: A brief review of current status and future prospects.

Small cell lung cancer (SCLC) often exhibits Rb deficiency, TRß and p130 deletion, and SKP2 amplification, suggesting TRß inactivation and SKP2 activation. It is reported that SKP2 targeted therapy is effective in some cancers in vitro and in vivo, but it is not reported for the treatment of SCLC and retinoblastoma. SKP2 is the synthetic lethal gene in SCLC and retinoblastoma, so SKP2 can be used for targeted therapy in SCLC and retinoblastoma. RB1 knockout mice develop several kinds of tumors, but Rb1 and SKP2 double knockout mice are healthy, suggesting that SKP2 targeted therapy may have significant effects on Rb deficient cancers with less side effects, and if successful in SCLC and retinoblastoma in vitro and in animal model, such compounds may be promising for the clinical treatment of SCLC, retinoblastoma, and variety of Rb deficient cancers. Previously our studies showed that retinoblastomas exhibit retinal cone precursor properties and depend on cone-specific thyroid hormone receptor ß2 (TRß2) and SKP2 signaling. In this study, we sought to suppress SCLC and retinoblastoma cell growth by SKP2 inhibitors as a prelude to targeted therapy in vitro and in vivo. We knocked down TRß2 and SKP2 or over-expressed p27 in SCLC and retinoblastoma cell lines to investigate SKP2 and p27 signaling alterations. The SCLC cell lines H209 as well as retinoblastoma cell lines Y79, WERI, and RB177 were treated with SKP2 inhibitor C1 at different concentrations, following which Western blotting, Immunostaining, and cell cycle kinetics studies were performed to study SKP2 and p27 expression ubiquitination, to determine impact on cell cycle regulation and growth inhibition. TRß2 knockdown in Y79, RB177 and H209 caused SKP2 downregulation and degradation, p27 up-regulation, and S phase arrest, whereas, SKP2 knockdown or p27 over-expression caused p27 accumulation and G1-S phase arrest. In the cell lines Y79, WERI, RB177, and H209 treatment with C1 caused SKP2 ubiquitination and degradation, p27 de-ubiquitination and accumulation, and cell growth arrest. SKP2 inhibitor C1 significantly suppressed retinoblastoma as well as SCLC cell growth by SKP2 degradation and p27 accumulation. In vivo study also showed inhibition of tumor growth with C1 treatment. Potential limitations of the success of such a therapeutic approach and its translational application in human primary tumors, and alternative approaches to overcome such limitations are briefly discussed for the treatment of retinoblastoma, SCLC and other RB-related cancers.

Tumor-associated retinal astrocytes promote retinoblastoma cell proliferation through production of IGFBP-5.

Retinoblastomas consist of cone-like neoplastic cells and diverse non-neoplastic cells whose roles in tumorigenesis have not been defined. Here, we investigated the glial cells that constitute 2% to 3% of the cells in retinoblastoma tumors, including their origin, their relationship to a potential retinoblastoma stem cell population, and their effects on tumor cell proliferation. Retinoblastoma glia consistently expressed the retinal astrocyte marker Pax2 but inconsistently expressed the Müller cell and occasional astrocyte marker CRALBP. Many of the glia expressed the stem cell-associated Sox2 but nevertheless were non-neoplastic as they coexpressed Rb and/or retained two RB1 alleles. Conversely, the glia were distinct from the non-neoplastic cells that strongly expressed the stem cell-associated ABCG2. Adherent Pax2(+),Sox2(+),Rb(+) glia readily grew from explanted retinoblastomas and produced soluble factors that enhanced the proliferation of cocultured retinoblastoma cells. This effect was emulated by normal retinal glia and appeared to be mediated by insulin-like growth factor binding protein-5 (IGFBP-5), as it was mimicked by recombinant IGFBP-5 and mitigated by neutralizing IGFBP-5 antibody. As glia-derived IGFBP-5 was earlier found to promote photoreceptor survival, our findings indicate that retinal astrocytes enhance the proliferation of cone-like retinoblastoma cells by deploying a factor that also provides trophic support to the tumor cells' non-neoplastic counterparts. These observations suggest that a tissue-specific microenvironmental feature cooperates with oncogenic mutations in a cancer cell of origin to promote retinoblastoma tumorigenesis.

Somatic Epigenetic Silencing of RIPK3 Inactivates Necroptosis and Contributes to Chemoresistance in Malignant Mesothelioma.

PURPOSE: Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma. EXPERIMENTAL DESIGN: RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and DNMT1 siRNA studies were performed to assess the mode of RIPK3 silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2'-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization. RESULTS: Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2'-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of RIPK3 expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of RIPK3 is associated with reduced RIPK3 expression and poor prognosis. CONCLUSIONS: These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of RIPK3 by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.

Distinction of conjunctival melanocytic nevi from melanomas by fluorescence in situ hybridization.

A conjunctival melanocytic nevus may on occasion be difficult to distinguish from melanoma both clinically and histopathologically. An unambiguous correct diagnosis is critical because of major differences in management and prognosis. We evaluated a fluorescence in situ hybridization (FISH) assay, which has previously been shown to be of value for the diagnosis of melanocytic nevi and melanomas of the skin, using probes targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1) and centromere 6 (CEP6), for its potential to assist in the distinction of conjunctival melanocytic nevi from melanomas. Four melanocytic nevi and eight melanomas of the conjunctiva were analyzed. Two of the melanomas were diagnostically problematic because of suboptimal histopathology. None of the conjunctival melanocytic nevi showed a level of chromosomal aberrations that met FISH criteria for a diagnosis of melanoma. All eight conjunctival melanomas (six unequivocal and two suspicious lesions) met FISH criteria for melanoma. Thus, results from FISH assay targeting 6p25, 6q23, 11q13 and centromere 6 correlated well with the histopathologic diagnoses and supported the histopathologic suspicion in two problem cases. The findings encourage further exploration of this technique as an ancillary method for the work-up of conjunctival melanocytic proliferations.

Cancer genomics of lung cancer including malignant mesothelioma: A brief overview of current status and future prospects.

Cancer as a genetic disease is by now well recognized. Genomic analysis of cancer cells, therefore, has greatly enhanced our ability to identify genetic alterations associated with various cancer types, including both lympho-hematopoietic as well as solid tumors. Chronic myeloid leukemia (CML), based on the specific diagnostic genetic abnormality has served as a prototype disease to clearly demonstrate the significance of the genomic analysis of cancer in identifying targeted therapy. Such a success has provided extra ordinary opportunities to investigate the role of genetic abnormalities and the pathways amenable to targeted therapy, not only in blood cancers but solid tumors such as Lung, Brain, Colon, Renal, Breast cancers as well as other epithelial and mesenchymal tumors. The main focus of this presentation is to illustrate the role of genomic analysis in targeting lung cancer, based on abnormalities or the pathways deregulated in tumor cells from individual patients. Lung cancer is one of the most common epithelial cancers associated with chronic inflammation due to cigarette smoking and other environmental carcinogens, and includes four distinct histologic type; non-small cell lung cancer (NSCLC); small cell lung cancer (SCLC) and squamous cell lung cancer. According to current estimates, 1.3 million cases of lung cancer are expected to be diagnosed worldwide annually, resulting in one million deaths. Since the discovery that patient's tumors with specific mutations in the EGFR may be sensitive to targeted therapeutic approach and the subsequent realization that the such mutations in the gene are not as prevalent, several cancer centers including ours initiated intense efforts to find other mutations or genomic alterations, which may serve as targets of specific therapy. Such efforts have successfully resulted in a battery of genes such as KRAS, ALK, C-MET, HER-2/neu, ROS1, etc., which have helped oncologists to triage the patients for personalized therapies. A significant proportion of patients with lung cancer, however, do not show any of the above genetic abnormalities. Approximately 90% of lung cancers exhibit RB1 mutation/deletion and or KRAS mutations, therefore, the signaling pathways, which regulate multistep tumorigenesis in lung cancer, are important for the treatment of histologic subtypes of lung cancer, which includes NSCLC & SCLC. Equally important was the findings that similar signaling pathways are also shared by other solid tumor types. We have investigated the role of these pathways to target these cancers and develop new strategies to treat lung, brain and related cancers. In addition, our translational studies in other tumor types such as NF2 related malignancies, specifically, Malignant Mesothelioma (MM), in which NF2 related pathway amenable to targeted therapies was identified. Selected examples representing experimental approaches will be discussed to illustrate the critical role of translational research in developing novel therapeutics for the successful and durable responses in some of these cancer types.

Feasibility of ex vivo FDG PET of the colon.

To facilitate future direct correlations between fluorine 18 fluorodeoxyglucose (FDG)-avid colonic lesions and immunohistochemical assay findings, the authors tested the feasibility of ex vivo FDG positron emission tomography (PET) of the colon resected from humans. In this institutional review board-approved, HIPAA-compliant study, the authors, after obtaining informed patient consent, injected FDG intraoperatively in five patients with neoplasms and imaged their resected colons approximately 3 hours later. The colon could be imaged during this fairly limited time interval, and polyps and cancers could be identified. No biologic tissue degradation occurred. The authors concluded that ex vivo FDG PET of the colon is feasible and, when combined with careful histologic and immunohistochemical analyses, may serve as a research tool to determine the mechanisms of the normal colonic uptake of FDG and the localization of FDG in polyps and cancers.

Reduced risk of secondary leukemia with fewer cycles of dose-intensive induction chemotherapy in patients with neuroblastoma.

BACKGROUND: We report a prospective study of secondary leukemia (SL)/myelodysplastic syndrome (MDS) in neuroblastoma (NB) patients treated with > or =5 cycles of dose-intensive chemotherapy. PROCEDURE: NB patients received induction with high-dose cyclophosphamide (4,200 mg/m(2))-doxorubicin (75 mg/m(2))-vincristine (cycles 1, 2, 4, 6, 8), and high-dose cisplatin (200 mg/m(2))-etoposide (600 mg/m(2)) (cycles 3, 5, 7). Bone marrow was examined every 1-3 months for > or =36 months, with inclusion of extensive chromosomal studies 1-3 months post-induction and 1-2x/year thereafter. RESULTS: One hundred eight four patients received 5 (n = 76), 6 (n = 45), 7 (n = 59), or 8 (n = 4) cycles. Eight patients developed SL/MDS (only one each in the 5- and 6-cycle groups), at 12-50 months, including two cases detected in surveillance studies. Among 108 patients who received > or =6 cycles, the 5-year cumulative incidence was 7.1% (95% CI: 2%, 12.2%), versus 0% among 54 patients who received 5 cycles without maintenance oral etoposide. Five-year cumulative incidences were 1.46%, 2.28%, and 8.47% among patients in the 5-, 6-, and 7-cycle groups, with fewer cycles having a significantly lower risk (P = 0.048). There was no significant association of risk with potentially leukemogenic consolidative treatments (targeted radiotherapy, myeloablative therapy, and oral etoposide). CONCLUSIONS: Reducing the number of dose-intensive cycles significantly decreases the risk of SL/MDS, yielding 5-year rates matching the low range (0.4-2.2%) reported for moderate-dose combination chemotherapy regimens used against other pediatric solid tumors.

Molecular characterization of permanent cell lines from primary, metastatic and recurrent malignant peripheral nerve sheath tumors (MPNST) with underlying neurofibromatosis-1.

Malignant peripheral nerve sheath tumors (MPNSTs) develop in patients with underlying NF1, and usually arise as a result of malignant transformation of a pre-existing plexiform neurofibroma. The clonal cytogenetic abnormalities reported in primary MPNST include complex karyotypes with chromosome numbers in the triploid or tetraploid range with recurrent abnormalities of several chromosomes including losses or imbalances. As a prelude to cell biological, pharmacological, and functional studies to investigate pathways and gene(s) associated with multistep tumorigenesis, which includes progression, metastasis and resistance to therapy in MPNST, detailed molecular cytogenetic and genetic analyses of cell lines from primary, metastatic and recurrent MPNST with underlying NF1 disorder have been performed. The clonal cytogenetic abnormalities detected in the primary tumor cell line were similar to those observed in primary cultures of this tumor. Due to the complexity of the rearrangements seen by G-banded karyotype analysis, further characterization of the clonal abnormalities in these three cell lines was performed by molecular cytogenetic techniques, including CGH and SKY. CGH analysis detected recurrent deletions of 9p, 12q21-q32, complete losses of the X-chromosome, and gains of the chromosomal segment 17q25 in all three cell lines. SKY analysis detected extensive clonal abnormalities in these cell lines. The nature and the alterations of the cell cycle regulators, particularly those associated with G1-S checkpoints and known to be deregulated in MPNST, were studied. These cell cycle regulators included those associated with Rb1-cyclin D1 and the p53 pathways. The findings are consistent with the argument that an imbalance between the cyclin activators of CDKs and inhibitory proteins such as p16 result in uncontrollable proliferation in the cell lines, associated with progression of the disease. LOH and expression of the p53 gene in metastatic and recurrent cell lines was observed, as reported by others. The role of biallelic inactivation of p53 gene in MPNST with underlying NF1 mutations, however, needs further study. Overexpression of Rb1-protein observed in metastatic and recurrent cell lines is indicative of its role in the progression of the disease. One of the most important observations of this study is that Nm23-H1 expression is closely associated with advanced or metastatic disease. In summary, MPNST cell lines derived from a patient with metastatic and recurrent disease with NF1 disorder were characterized and a gene associated with metastatic potential which is amenable to therapeutic and chemo-preventative approaches was identified. These cell lines with extensive characterization of genetic abnormalities are likely to provide important reagents for biochemical, molecular and pharmacological studies related to MPNST.

Transplantation in remission improves the disease-free survival of patients with advanced myelodysplastic syndromes treated with myeloablative T cell-depleted stem cell transplants from HLA-identical siblings.

From 1985 to 2004, 49 patients with advanced myelodysplastic syndromes (MDS) (> or =5% blasts) or acute myeloid leukemia (AML) transformed from MDS underwent T cell depleted bone marrow or peripheral blood hematopoietic stem cell transplantation (HSCT) from HLA-identical siblings following conditioning with a myeloablative regimen that included total body irradiation (44 patients) or busulfan (5 patients). Thirty-six patients received chemotherapy (3 low dose and 33 induction doses) before conditioning, and 13 patients did not receive any chemotherapy. Prior to transplantation, 22 of the 36 treated patients were in hematologic remission; 4 were in a second refractory cytopenia phase (26 responders); 8 had failed to achieve remission; and 2 of the responders had progression or relapse of their MDS (10 failures). No post-transplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD) was given. The median age was 48 yrs (range 13-61). Forty-five of the 49 patients engrafted; 2 had primary graft failure; and 2 died before engraftment. Only 3 patients developed acute GVHD (aGVHD) (grades I and III) and 1 chronic GVHD (cGVHD). At 3 yrs post-transplantation, the overall survival (OS) was 54% in the responders; 31% in the untreated group; and 0% in the failure group (P=.0004). The disease free survival (DFS) was 50%, 15% and 0% in each group respectively (P=.0008). In multivariate analysis, disease status before cytoreduction remained highly correlated with DFS (P<.001). The cumulative incidence (CI) of relapse at 2-yrs post-transplantation for the responders was 23%; for the untreated group was 38%; and for the failures was 50%. The CI of non-relapse mortality at 2-yrs post-transplantation, for the responders was 23%; for the untreated group was 38%; and for the failures was 40%. All survivors achieved a Karnofsky Performance Status (KPS) of > or =90. These results indicate that patients with advanced MDS who achieve and remain in remission or a second refractory cytopenia phase with chemotherapy before conditioning can achieve successful long-term remissions following a myeloablative T cell depleted allogeneic HSCT.

Teratocarcinosarcoma of the nasal cavity and paranasal sinuses: report of 3 cases with assessment for chromosome 12p status.

Sinonasal teratocarcinosarcoma (SNTCS) is a rare malignant neoplasm with 63 reported cases to date. Patients are exclusively adults, with a mean age of 60 years and marked male predominance. Histologically, these tumors are characterized by the presence of admixed epithelial and mesenchymal components. The histogenesis of SNTCS remains uncertain and genetic studies have not been reported to date. Two SNTCSs from the archives of Memorial Sloan-Kettering Cancer Center and one submitted from St Luke's-Roosevelt Hospital Center were evaluated by fluorescent in situ hybridization for amplification of chromosome12p, an event usually associated with the genesis of bona fide germ cell neoplasms (including mediastinal and testicular teratomas). Microscopic examination revealed admixed epithelial and mesenchymal elements in all 3 cases; benign squamous and glandular epithelium and neuroepithelial tissue were identified, the squamous epithelium demonstrating "fetal-like" cytoplasmic clearing. Mesenchymal proliferations were recognized ranging from well-differentiated smooth muscle to high-grade sarcoma. A malignant germ cell component was not identified in any of the cases. Fluorescent in situ hybridization evaluation demonstrated only 2 copies of chromosome 12 per case. Although the histogenesis of SNTCS remains uncertain, we have found an absence of 12p amplification in 3 cases. Our findings suggest that 12p amplification, if it occurs at all in this setting, is exceptional and that SNTCS is a somatic-type neoplasm exhibiting divergent differentiation rather than a germ cell tumor.

A mouse model recapitulating molecular features of human mesothelioma.

Malignant mesothelioma has been linked to asbestos exposure and generally has a poor prognosis because it is often diagnosed in advanced stages and is refractory to conventional therapy. Human malignant mesotheliomas accumulate multiple somatic genetic alterations, including inactivation of the NF2 and CDKN2A/ARF tumor suppressor genes. To better understand the significance of NF2 inactivation in malignant mesothelioma and identify tumor suppressor gene alterations that cooperate with NF2 loss of function in malignant mesothelioma pathogenesis, we treated Nf2 (+/-) knockout mice with asbestos to induce malignant mesotheliomas. Asbestos-exposed Nf2 (+/-) mice exhibited markedly accelerated malignant mesothelioma tumor formation compared with asbestos-treated wild-type (WT) littermates. Loss of the WT Nf2 allele, leading to biallelic inactivation, was observed in all nine asbestos-induced malignant mesotheliomas from Nf2 (+/-) mice and in 50% of malignant mesotheliomas from asbestos-exposed WT mice. For a detailed comparison with the murine model, DNA analyses were also done on a series of human malignant mesothelioma samples. Remarkably, similar to human malignant mesotheliomas, tumors from Nf2 (+/-) mice showed frequent homologous deletions of the Cdkn2a/Arf locus and adjacent Cdkn2b tumor suppressor gene, as well as reciprocal inactivation of Tp53 in a subset of tumors that retained the Arf locus. As in the human disease counterpart, malignant mesotheliomas from the Nf2 (+/-) mice also showed frequent activation of Akt kinase, which plays a central role in tumorigenesis and therapeutic resistance. Thus, this murine model of environmental carcinogenesis faithfully recapitulates many of the molecular features of human malignant mesothelioma and has significant implications for the further characterization of malignant mesothelioma pathogenesis and preclinical testing of novel therapeutic modalities.

Genomic instability and proliferation/survival pathways in RB1-deficient malignancies.

Genomic instability (GIN) is a hallmark of most cancer cells. However, compared to most human cancer cell types, the retinoblastoma tumor cells show a relatively stable genome. The fundamental basis of this genomic stability has yet to be elucidated, and the role of certain proteins involved in cell cycle regulation may be the key to the development of these specific genotypes. We examined whether thyroid hormone receptor beta 1 and 2 (TRß1 and TRß2), known to regulate tumorigenesis, and PTTG1, a mitotic checkpoint protein, play a role in maintaining genomic stability in retinoblastoma. In order to elucidate the role of these proteins in development of aneuploidy/polyploidy, an indicator of GIN, we first studied comparative GIN in retinoblastomas and multiple RB mutant cancer cell lines using single nucleotide polymorphism (SNP) analysis. We then utilized pLKO lentiviral vectors to selectively modify expression of the targeted cell cycle proteins and interpret their effect on downstream cell cycle proteins and their relative effects on the development of polyploidy in multiple tumor cell lines. The SNP analysis showed that retinoblastomas displayed relatively fewer genomic copy number changes as compared to other RB1-deficient cancer cell lines. Both TRß1 and TRß2 knockdown led to accumulation of E2F1 and PTTG1 and increased GIN as demonstrated by an increase in polyploidy. Downregulation of PTTG1 led to a relative decrease in GIN while upregulation of PTTG1 led to a relative increase in GIN. Knockdown of E2F1 led to a downstream decrease in PTTG1 expression. Rb-knockdown also upregulated E2F1 and PTTG1 leading to increased GIN. We showed that Rb is necessary for PTTG1 inhibition and genomic stability. A relatively stable genome in retinoblastoma tumor cells is maintained by TRß1 and TRß2-mediated PTTG1 inhibition, counteracting Rb-deficiency-related GIN. TRß1, TRß2 and Rb-KD all led to the downstream PTTG1 accumulation, apparently through an activation of E2F1 resulting in extensive genomic instability as seen in other Rb-deficient tumors.

Gain of chromosome 1q portends worse prognosis in multiple myeloma despite novel agent-based induction regimens and autologous transplantation.

We aimed to identify whether the use of autologous hematopoietic cell transplantation (HCT) impacts outcomes for multiple myeloma patients with gains of chromosome 1q (+1q). We retrospectively identified 95 patients, 21% having +1q. For patients with +1q, the overall response rate to induction was 85%, with 40% having ≥ VGPR and 20% achieving a CR, similar to non +1q patients (p = .64). The median PFS from diagnosis with +1q was 2.1 years (95% CI: 1.2-not reached (NR)) vs 4.3 years (95% CI: 3.3 yrs-NR) without +1q (p = .003). Median OS from diagnosis was 4.4 years (95% CI: 2.9-NR) vs not reached, respectively (p = .005). On molecular analysis using the Foundation One Heme assay, the most common mutations seen in +1q patients included TP53 (38%) and KRAS (25%). Overall, gain of 1q portends worse PFS and OS which was not negated by auto HCT. Such patients will likely require additional therapy to improve their survival.

SKP2 Activation by Thyroid Hormone Receptor ß2 Bypasses Rb-Dependent Proliferation in Rb-Deficient Cells.

Germline RB1 mutations strongly predispose humans to cone precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRß2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRß1. TRß2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRß2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRß1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRß2 counteracts TRß1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838-50. ©2017 AACR.

Human and mouse mesotheliomas exhibit elevated AKT/PKB activity, which can be targeted pharmacologically to inhibit tumor cell growth.

Malignant mesotheliomas (MMs) are very aggressive tumors that respond poorly to standard chemotherapeutic approaches. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in tumor aggressiveness, in part by mediating cell survival and reducing sensitivity to chemotherapy. Using antibodies recognizing the phosphorylated/activated form of AKT kinases, we observed elevated phospho-AKT staining in 17 of 26 (65%) human MM specimens. In addition, AKT phosphorylation was consistently observed in MMs arising in asbestos-treated mice and in MM cell xenografts. Consistent with reports implicating hepatocyte growth factor (HGF)/Met receptor signaling in MM, all 14 human and murine MM cell lines had HGF-inducible AKT activity. One of nine human MM cell lines had elevated AKT activity under serum-starvation conditions, which was associated with a homozygous deletion of PTEN, the first reported in MM. Treatment of this cell line with the mTOR inhibitor rapamycin resulted in growth arrest in G1 phase. Treatment of MM cells with the PI3K inhibitor LY294002 in combination with cisplatin had greater efficacy in inhibiting cell proliferation and inducing apoptosis than either agent alone. Collectively, these data indicate that MMs frequently express elevated AKT activity, which may be targeted pharmacologically to enhance chemotherapeutic efficacy. These findings also suggest that mouse models of MM may be useful for future preclinical studies of pharmaceuticals targeting the PI3K/AKT pathway.

Growth inhibition and induction of apoptosis in mesothelioma cells by selenium and dependence on selenoprotein SEP15 genotype.

Malignant mesotheliomas (MMs) are aggressive tumors derived from mesothelial cells lining the lungs, pericardium and peritoneum, and are often associated with occupational asbestos exposure. Suppression subtractive hybridization was used to identify genes differentially expressed in MM cells compared to normal mesothelial cells. A gene, SEP15, encoding a 15-kDa selenium-containing protein was isolated using this approach and was subsequently shown to be downregulated in approximately 60% of MM cell lines and tumor specimens. A SEP15 polymorphic variant, 1125A, resides in the SECIS recognition element in the 3'-UTR and may influence the efficiency of Sec incorporation into the protein during translation. Since previous studies have implicated a potential role of the trace element selenium as a chemopreventive agent in animal models and in several types of human cancer, we investigated the effect of selenium on MM cells and its dependence on SEP15 genotype. Selenium was shown to inhibit cell growth and induce apoptosis in a dose-dependent manner in MM cells but had minimal effect on normal mesothelial cells. However, MM cells with downregulated SEP15 or the 1125A variant were somewhat less responsive to the growth inhibitory and apoptotic effects of selenium than MM cells expressing wild-type protein. RNAi-based knockdown studies demonstrated that SEP15 inhibition makes sensitive MM cells more resistant to selenium. These data imply that selenium may be useful as a chemopreventive agent in individuals at high risk of MM due to asbestos exposure, although those with the 1125A polymorphism may be less responsive to the protective benefits of dietary selenium supplementation.