Wild-type Rb Research Articles

Comprehensive genomic profiles of small cell lung cancer.

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Abstract B115: PD0332991 induces epithelial to mesenchymal transition in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains the fourth leading cause of cancer-related death among both men and women in the United States, with an overall survival rate of 6%. Moreover, the median survival in PDAC is only five to six months, underscoring the need for improved therapies. Cancer cell proliferation and cell cycle dysregulation is a major target in cancer treatment. Cyclin-dependent kinase (CDK) and cyclin inhibitors act as anti-proliferative agents that target upregulated cell-cycle progression proteins in various tumors. The efficacy of PD0332991, a CDK4/6 inhibitor, is currently being investigated in clinical trials in patients with retinoblastoma protein (Rb)-positive breast, ovarian, and non-small-cell lung cancers, but has not yet been examined for treatment in PDAC. We have recently shown that PD0332991 inhibits cell proliferation in PDAC cell lines but initiates epithelial to mesenchymal transition (EMT) and induces cancer cell invasion in some of these cells. Using human PDAC tissues and tissues from genetically engineered mouse models (GEMMs) of PDAC, we also recently showed that loss of RB function is common in PDAC. To further assess the therapeutic potential of PD0332991 in PDAC, we now compared its effects in PANC-1 human PDAC cells (wild-type RB1 gene) and in murine PDAC cells devoid of RB that were derived from a GEMM expressing mutant KRASG12D (KRC mice). In PANC-1 cells, PD0332991 (1 μM) inhibited proliferation and induced EMT. By contrast, in KRC 1022-4 murine PDAC cells, the same concentration of PD0332991 induced EMT without altering proliferation. Moreover, removal of PD0332991 from the incubation medium reversed its growth inhibitory actions in PANC-1 cells, but did not reverse its effects on EMT in either PANC-1 or KRC 1022-4 cells. Taken together, these findings suggest that PD0332991 may exert long-term deleterious effects in patients with PDAC limiting its potential therapeutic use in this deadly cancer, except in conjunction with agents that target EMT. This study was supported, in part, by NCI grant R37-075059 to MK. Citation Format: I. E. Imasuen, A. Jesse Gore, M. Korc. PD0332991 induces epithelial to mesenchymal transition in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B115.

Abstract 4402: Synergistic cell death in nedd8 and hrb27c mutant cells under an rbf-negative background is dependent on EGFR/MAPK signaling

Abstract The Rb (retinoblastoma) protein is highly functionally conserved between the Drosophila, and deregulation and mutations in the Rb pathway are common events across a wide range of cancers. Here, we identify two novel synthetic lethal interactions between Rb and mutant alleles of Nedd8 and Hrb27c that were identified through an unbiased genetic screen in Drosophila that resulted in apoptosis in both the wing disc pouch and the region of the eye disc posterior to the morphogenetic furrow. However, the molecular mechanisms behind the apoptotic phenotypes in both synthetic lethal double mutants have yet to be elucidated. It is known that wild-type Rb normally sequesters and can prevent the E2F1 transcription factor from promoting transcription of hid, the major pro-apoptotic regulator in imaginal discs. Synthetic lethality is therefore likely to be contingent on EGFR/hid signaling, and I will determine if such is the case through a series of genetic epistasis tests. Interestingly, preliminary evidence also suggests that EGFR/MAPK signaling may be ablated in both mutants of Nedd8 and Hrb27c. However, this observation is completely novel and therefore the molecular mechanisms behind downregulation of MAPK signaling have yet to be elucidated in both these mutants. I will again perform a series of epistasis experiments to determine how Nedd8 and Hrb27c mutations mechanistically affect EGFR/MAPK signaling. This will be achieved first by determining if signaling is downregulated either during EGFR ligand processing or downstream of the EGFR receptor. This will be followed by subsequent experiments to determine which specific element in either pathway is being affected by Nedd8 or Hrb27c ablation. I therefore hypothesize that inhibition of both nedd8 and hrb27c synergistically induce cell death in Rb-negative cells in a process that is dependent on both E2F1 activity and downregulation of MAPK signaling. Many existing treatment regimens fail to take into account the unique biological properties of cancer cells compared to their normal counterparts, thus often resulting in off-target effects and limitations in dosage. This proposal is therefore both novel and significant because the mechanisms behind both the Rb/Hrb27c and Rb/Nedd8 synthetic lethal interactions have yet to be elucidated and characterization of such interactions such as these have the potential to catalyze the development of new therapeutic strategies in the treatment of cancer. Note: This abstract was not presented at the meeting. Citation Format: Robin Zhang, Tianyi Zhang, Zhentao Sheng, Wei Du. Synergistic cell death in nedd8 and hrb27c mutant cells under an rbf-negative background is dependent on EGFR/MAPK signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4402. doi:10.1158/1538-7445.AM2014-4402

Reduced expression of the retinoblastoma protein shows that the related signaling pathway is essential for mediating the antineoplastic activity of erufosine.

Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G2 phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G2 arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.

Abstract C12: Molecular mechanisms of aggressiveness of Rb-deficient tumors

Abstract Osteosarcomas (OS) and small cell lung carcinomas (SCLC) are poorly differentiated, highly aggressive tumors, with an early proclivity to metastasize. Prognosis is poor for both, with only 10-20% of patients achieving long-term disease-free intervals. Still, OS and SCLC aggressivity remain to be molecularly explained. Interestingly, these cancer types show high rates of inactivation of the RB1 gene coding for the retinoblastoma protein (Rb), a known cell cycle repressor. This observation led us to hypothesize that in OS and SCLC, Rb loss during early tumorigenesis facilitates both increased proliferation and an early tendency for metastasis. Supporting our hypothesis, our previous studies in osteoblasts showed that Rb transcriptionally regulates a wide repertoire of cell adhesion genes, including those coding for adherens junction cadherins and integrins. We also found that adhrens junction assembly requires the Rb-dependent transcriptional repression of Pak1, a Rac1-binding kinase that destabilizes cell adhesion when up-regulated. Thus, the early proclivity for metastasis of OS and SCLC may be related to perturbations in cell adhesion due to Rb loss. Here we present new data using the SCLC line H187 and the non-small cell lung carcinoma (NSCLC) cell lines H1975 and H520. SCLC carcinomas have high rates (> 90%) of Rb loss while NSCLC are known to be Rb wild type. Therefore, comparing H1975 and H520 vs. H187 provided us with an Rb-proficient vs. Rb-deficient system similar to the one we previously used to study Rb-engendered effects in osteoblasts. Our immunoblots and qRT-PCR showed that the Rb-null H187 cells expressed dramatically reduced levels of E-cadherin mRNA and protein relative to H1975 and H520, confirming that Rb loss impairs cell adhesion due to adherens junction loss. Interestingly, N-cadherin is up-regulated in H187 cells, suggesting that Rb loss triggers a cadherin switch similar to the one associated with epithelial-to-mesenchymal transitions. Other cell adhesion-related genes we found down-regulated in Rb-null H187 cells relative to H1975 and H520, as determined by qRT-PCR, were α-catenin, β-catenin, OB-cadherin, and the cytoskeletal adapter proteins eplin, vinculin, formin-1, and α-actinin. Our immunoblots also showed decreased levels of merlin, an adherens junction-interacting protein, in H187 cells. We also studied the expression of Rac1-binding proteins, given their involvement in cell adhesion. Our immunoblots and qRT-PCR showed reduced levels of IQGAP1 in H187 cells relative to H1975 and H520 cells. Given that IQGAP1 is a GTPase activating protein (GAP) that represses Rac1 by promoting its GDP-bound inactive state, we postulate that Rb represses Rac1 function via IQGAP1. Beta-2-chimaerin, another Rac1 GAP was also down regulated at the protein level in H187 cells. Taken together, our data in lung cell lines recapitulate most of our findings in osteoblasts, both in regards to the effect of Rb on cell adhesion and to the need for the Rb-mediated repression of Rac1 for the establishment of cell adhesion. Based on our data, we propose that Rb loss in OS and SCLC, not only leads to an increased proliferative capacity during early tumorigenesis, but also exacerbates aggressiveness of tumors at early stages by perturbing cellular adhesion, which in turn facilitates tumor cell detachment and metastasis. Citation Format: Jonathan González-Flores, Ruth Cruz-Cosme, Yariana Rodríguez-Ortiz, Sarah Brady, Ricardo Fraticelli-Rosado, Brienne Engel, Douglas Cress, Pedro G. Santiago-Cardona. Molecular mechanisms of aggressiveness of Rb-deficient tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C12.

The Role of Tumor Suppressor Dysregulation in Prostate Cancer Progression

Androgen receptor activity is essential for prostate cancer development and progression. While there are classically defined roles for the retinoblastoma (Rb) and p53 tumor suppressor pathways in maintenance of cell cycle control and the DNA damage response, recent studies have demonstrated a direct role of these two pathways in regulating AR expression and function. While the role of Pten deregulation in prostate cancer has provided much insight in to the mechanisms underlying prostate cancer initiation and progression, emerging roles for Rb and p53 are likely to further expand upon our understanding of tumor suppressor/nuclear receptor interaction. As disconnecting mitogenic signaling from AR-mediated gene transcription underlies the progression to castrate resistant prostate cancer (CRPC), functional inactivation of these two tumor suppressor pathways represents one mechanism through which AR protein levels can be upregulated and AR-mediated gene transcription can become aberrant. Importantly, recent advances in small molecule inhibitor design and discovery have led to the identification of agents capable of targeting these two prominent pathways and restoring the function of deregulated wild-type Rb and p53 protein. While such agents have undergone extensive study in many solid tumor types, the additional importance of Rb and p53 in restraining transcription of the AR gene within the prostate provides impetus for examining how loss of these two tumor suppressor proteins can facilitate transition of prostate cancers to CRPC. As will be reviewed in this article, restoration of Rb and p53 functions are not only important in regard to shortterm cell cycle regulation and response to genomic stresses, but likely have direct implications for deregulation of the AR locus.

The cooperative effect of p53 and Rb in local nanotherapy in a rabbit VX2 model of hepatocellular carcinoma

Background/aimA local nanotherapy (LNT) combining the therapeutic efficacy of trans-arterial embolization, nanoparticles, and p53 gene therapy has been previously presented. The study presented here aimed to further improve the incomplete tumor eradication and limited survival enhancement and to elucidate the molecular mechanism of the LNT.MethodsIn a tumor-targeting manner, recombinant expressing plasmids harboring wild-type p53 and Rb were either co-transferred or transferred separately to rabbit hepatic VX2 tumors in a poly-L-lysine-modified hydroxyapatite nanoparticle nanoplex and Lipiodol® (Guerbet, Villepinte, France) emulsion via the hepatic artery. Subsequent co-expression of p53 and Rb proteins within the treated tumors was investigated by Western blotting and in situ analysis by laser-scanning confocal microscopy. The therapeutic effect was evaluated by the tumor growth velocity, apoptosis and necrosis rates, their sensitivity to Adriamycin® (ADM), mitomycin C, and fluorouracil, the microvessel density of tumor tissue, and the survival time of animals. Eventually, real-time polymerase chain reaction and enhanced chemiluminescence Western blotting were used to investigate the expressive changes of important genes related to the therapy.ResultsThe administration procedure proved safe for the rabbits’ liver function, the p53 plus Rb LNT showed significantly better antitumoral effect and lower expression of malignant genes than the p53 or Rb LNT, although no significant difference was observed in animal survival when the p53 plus Rb LNT was compared with the p53 LNT.ConclusionRb works synergistically with p53 in combined therapy mediated by a poly-L-lysine-modified hydroxyapatite nanoparticle nanoplex to augment the antitumoral effect through the downregulated expression of important genes related to apoptosis, necrosis, growth, differentiation and multidrug resistance of tumor cells. LNT with p53 and Rb is potentially an effective antitumor therapy for hepatocellular carcinoma.

Retinoblastoma gene‐independent G1 phase arrest by flavone, phosphatidylinositol 3‐kinase inhibitor, and histone deacetylase inhibitor

In most human malignant tumors, retinoblastoma tumor-suppressor gene (RB) product is inactivated by phosphorylation. Therefore, cancer preventive agents or molecular-targeting agents can inhibit the tumor growth at G(1) phase through RB reactivation. However, little is known about the effectiveness of RB reactivating agents against malignancies with mutated RB. We report here that chemopreventive agent flavone, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) also induce G(1) phase arrest in malignant tumor cells with mutated RB. In human prostate cancer DU145 cells with mutated RB, flavone increased cyclin-dependent kinase (CDK) inhibitors p21 and p27, and reduced cdk4 and cdk6, resulting in decrement of phosphorylated RB family proteins p130 and p107. LY294002 also dephosphorylated p107 and p130 proteins, whereas TSA dephosphorylated p130, but not p107. Furthermore, flavone induced G(1) phase arrest in both mouse embryo fibroblast (MEF) wild-type and MEF RB(-/-) cells, but did not do so in RB, p107, and p130 triple-knockout MEF cells. These results suggested that p130 and p107 contributed to G(1) phase arrest by flavone in RB-mutated cells. However, flavone induced tumor suppressor microRNA miR-34a with reduction of E2F1 and E2F3, known to be downregulated by miR-34a, raising the possibility that miR-34a might partially contribute to G(1) arrest by flavone. These results raise the possibility that RB reactivating chemopreventive agents or molecular targeting agents might also be effective against a variety of malignant tumor cells with mutant RB.

P16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.

The effects of retinoblastoma (Rb) deletion on intestinal lipid absorption

BackgroundThe Retinoblastoma protein (Rb) may play a role in modulating the absorption of lipids within the small bowel. Rb null mice are characterized by an increased intestinal surface area compared to wild‐type (WT) mice. This study investigates the effects of Rb deficiency on lipid absorptive capacity with normal and high fat diets.MethodsRb null and WT mice were maintained on normal and high fat diets for short and long–term periods. Mice where weighted weekly. Fat absorption and fecal fat were determined using an extraction protocol. Lean mass and body fat were determined via nuclear magnetic resonanceResultsNo differences in weight were found between Rb null and WT mice. The Rb null mice on normal chow had higher fecal fat compared to WT; however no significant difference in fat absorption was seen between the groups. Similar results were observed after the same mice were placed on a short‐term high fat diet. An extended high fat diet showed Rb null mice maintained a higher fecal fat while fat absorption increased in WT mice. No significant differences in body fat or lean mass were found between Rb null and WT mice on a prolonged high fat diet.ConclusionRegardless of the type of diet given, Rb null mice absorb lipids at a lower capacity compared to WT mice. Enhanced bowel surface area seems to serves no advantage in regards to lipid absorption.Research supported by NIH grant DK 59288‐06A2

Preclinical in vitro and in vivo evaluation of the potent and specific cyclin-dependent kinase 2 inhibitor NU6102 and a water soluble prodrug NU6301

To facilitate the evaluation of CDK2 (cyclin-dependent kinase 2) as a cancer target, the in vitro and in vivo properties of NU6102 (O6-cyclohexylmethyl-2-(4′-sulphamoylanilino)purine) and a water soluble prodrug (NU6301) were investigated. NU6102 selectively inhibited the growth of CDK2 WT (wild type) versus KO MEFs (knockout mouse embryo fibroblasts) (GI50 (concentration required to inhibit cell growth by 50%) 14μM versus >30μM), and was more growth-inhibitory in p53 mutant or null versus p53 WT cells (p=0.02), and in Rb (retinoblastoma protein) WT SKUT-1B versus SKUT 1 Rb deficient cells (p=0.01). In SKUT-1B cells NU6102 induced a G2 arrest, inhibition of Rb phosphorylation and cytotoxicity (LC50 2.6μM for a 24h exposure). The prodrug NU6301 rapidly generated NU6102 in vitro in mouse plasma, and tumour NU6102 levels in vivo consistent with activity in vitro. Eight or 12hourly dosing of 120mg/kg NU6301 for 10days was well tolerated in SKUT-1B tumour-bearing mice and inhibited Rb phosphorylation in tumour tissue. Two (8hourly dosing) and 3 (12hourly dosing) day tumour growth delay was observed (p=0.04 and p=0.007, respectively) following NU6301 administration. NU6102 and its prodrug NU6301 have pharmacological properties consistent with CDK2 inhibition, and represent useful tool molecules for the evaluation of CDK2 as a target in cancer.

Abstract 2716: Overexpression of p16INK4a in Merkel cell carcinoma

Abstract Merkel cell polyomavirus (MCV) is a recently discovered human oncogenic virus. The majority of Merkel cell carcinomas (MCC) have been found to be infected with MCV. The MCV large T antigen (LTA) consists of an Rb binding motif that is functionally analogous to the E7 protein of high-risk human papillomavirus (HPV) types (eg. 16 and 18). It has been well documented that inactivation of Rb leads to overexpression of p16INK4a. Clinically, immunohistochemistry for p16INK4a has proven useful in the diagnosis of HPV driven epithelial malignancies including cervical as well as head and neck cancers. The present study explores the relationship between MCV infection and p16INK4a expression using an in vitro cell culture system and 10 clinical specimens of MCC that have been diagnosed at the San Diego VA Hospital over a 21 year period. First, IMR-90 cells were transfected with MCV LTA plasmids, which carry either wild-type or mutant Rb binding motif for immunofluorescence analysis. Only cells transfected with wild-type, but not mutant LTA, were associated with p16INK4a up-regulation. Furthermore, a strong positive correlation was established between MCV LTA and p16INK4a expression in formalin-fixed, paraffin-embedded MCC samples by immunohistochemical staining using p16INK4a and MCV LTA specific monoclonal antibodies, and real-time PCR to detect MCV with two pairs of primers. In conclusion, strong association between p16INK4a expression and MCC suggests that the MCV and HPV related carcinomas share some common oncogenic pathways. In addition, p16INK4a positivity can be a useful diagnostic tool in differentiating MCC from other small blue cell tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2716. doi:10.1158/1538-7445.AM2011-2716

MiR-449a causes Rb-dependent cell cycle arrest and senescence in prostate cancer cells

MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) that regulate gene expression by repressing translation or triggering the degradation of complementary mRNA sequences. Certain miRNAs have been shown to function as integral components of the p53 and/or retinoblastoma (Rb) regulatory networks. As such, miRNA dysregulation can have a profound effect on cancer development. Previous studies have shown that miR-449a is down-regulated in human prostate cancer tissue and possesses potential tumor suppressor function. In the present study, we identify miR-449a-mediated growth arrest in prostate cancer cells is dependent on the Rb protein. We show that mutant Rb prostate cancer cells (DU- 145) are resistant to cell cycle arrest and cellular senescence induced by miR- 449a, while overexpression of wild-type Rb in DU-145 sublines (DU-1.1 and B5) restores miR-449a function. In silico analysis of 3'UTR regions reveal a putative miR-449a target site in the transcript of Cyclin D1 (CCND1); an oncogene involved in directly regulating Rb activity and cell cycle progression. Luciferase 3'UTR reporter constructs and inhibitory oligonucleotides confirm that Cyclin D1 is a direct downstream target of miR-449a. We also reveal that miR-449a suppresses Rb phosphorylation through the knockdown of Cyclin D1 and previously validated target HDAC1. By targeting genes involved in controlling Rb activity, miR- 449a regulates growth and senescence in an Rb-dependent manner. These data indicate that miR-449a is a miRNA component of the Rb pathway and its tumor suppressor-like effects, in part, depends on Rb status in prostate cancer cells.

Rb inactivation accelerates neoplastic growth and substitutes for recurrent amplification of cIAP1, cIAP2 and Yap1 in sporadic mammary carcinoma associated with p53 deficiency

Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis. We used newly generated MMTV-Cre105Ayn mice to inactivate p53 and/or Rb strictly in the mammary epithelium and to determine recurrent genomic changes associated with deficiencies of these genes. p53 inactivation led to formation of estrogen receptor positive raloxifene-responsive mammary carcinomas with features of luminal subtype B. Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation. Genome-wide analysis of mammary carcinomas identified a recurrent amplification at chromosome band 9A1, a locus orthologous to human 11q22, which contains protooncogenes cIAP1 (Birc2), cIAP2 (Birc3) and Yap1. Interestingly, this amplicon was preferentially detected in carcinomas carrying wild-type Rb. However, all three genes were overexpressed in carcinomas with p53 and Rb inactivation, likely due to E2F-mediated transactivation, and cooperated in carcinogenesis according to gene knockdown experiments. These findings establish a model of luminal subtype B mammary carcinoma, identify critical role of cIAP1, cIAP2 and Yap co-expression in mammary carcinogenesis and provide an explanation for the lack of recurrent amplifications of cIAP1, cIAP2 and Yap1 in some tumors with frequent Rb-deficiency, such as mammary carcinoma.

Counterbalance between RB inactivation and miR-17–92 overexpression in reactive oxygen species and DNA damage induction in lung cancers

Small-cell lung cancer (SCLC) is a highly aggressive disease that exhibits rapid growth and genetic instability. We found earlier frequent overexpression of the miR-17-92 microRNA cluster, and showed that SCLC cells were addicted to continued expressions of miR-17-5p and miR-20a, major components of this microRNA cluster. In this study, we identified the frequent presence of constitutively phosphorylated H2AX (gamma-H2AX), which reflects continuing DNA damage, preferentially in SCLC. Knockdown of RB induced gamma-H2AX foci formation in non-small cell lung cancer (NSCLC) cells with wild-type RB, in association with growth inhibition and reactive oxygen species (ROS) generation, which was canceled by overexpression of miR-17-92. Conversely, induction of gamma-H2AX was observed in a miR-17-92-overexpressing SCLC cell line with miR-20a antisense oligonucleotides. These findings suggest that miR-17-92 overexpression may serve as a fine-tuning influence to counterbalance the generation of DNA damage in RB-inactivated SCLC cells, thus reducing excessive DNA damage to a tolerable level and consequently leading to genetic instability. Therefore, miR-17-92 may be an excellent therapeutic target candidate to elicit excessive DNA damage in combination with DNA-damaging chemotherapeutics.

Haploinsufficiency of the retinoblastoma protein gene reduces diet-induced obesity, insulin resistance, and hepatosteatosis in mice

Brown adipose tissue activity dissipates energy as heat, and there is evidence that lack of the retinoblastoma protein (pRb) may favor the development of the brown adipocyte phenotype in adipose cells. In this work we assessed the impact of germ line haploinsufficiency of the pRb gene (Rb) on the response to high-fat diet feeding in mice. Rb(+/-) mice had body weight and adiposity indistinguishable from that of wild-type (Rb(+/+)) littermates when maintained on a standard diet, yet they gained less body weight and body fat after long-term high-fat diet feeding coupled with reduced feed efficiency and increased rectal temperature. Rb haploinsufficiency ameliorated insulin resistance and hepatosteatosis after high-fat diet in male mice, in which these disturbances were more marked than in females. Compared with wild-type littermates, Rb(+/-) mice fed a high-fat diet displayed higher expression of peroxisome proliferator-activated receptor (PPAR)gamma as well as of genes involved in mitochondrial function, cAMP sensitivity, brown adipocyte determination, and tissue vascularization in white adipose tissue depots. Furthermore, Rb(+/-) mice exhibited signs of enhanced activation of brown adipose tissue and higher expression levels of PPARalpha in liver and of PPARdelta in skeletal muscle, suggestive of an increased capability for fatty acid oxidation in these tissues. These findings support a role for pRb in modulating whole body energy metabolism and the plasticity of the adipose tissues in vivo and constitute first evidence that partial deficiency in the Rb gene protects against the development of obesity and associated metabolic disturbances.

On the dynamics of breast tumor development in women carrying germline BRCA1 and BRCA2 mutations

We used mathematical models to analyze the age-incidence curve of breast carcinoma for individuals carrying a germline mutation in the BRCA1 or BRCA2 gene locus. Although many genomic abnormalities have been identified in breast tumors, we found that a two-stage model fit the data well. A one-hit model was not, however, consistent with the data. The results supported the hypothesis that the first hit represents loss of the wild type BRCA1 or BRCA2 allele as this occurs at a rate very similar to that for loss of the wild-type RB allele in retinoblastoma. Loss of the wild-type BRCA1 or BRCA2 allele appears to destabilize the genome as the second event occurs at a much higher rate. The second event is "rate limiting" in the sense that its occurrence is constrained by the limited number of intermediate cells with doubly mutated BRCA1 or BRCA2 alleles. The second event may not be unique, however. Loss of the wild-type BRCA allele appears to result in an increased rate for subsequent genomic events. A second event increasing proliferation of the partially malignant intermediate clone may lead inexorably to production and selection of cells with additional mutations in genes that facilitate tumor progression.

Coordinate Interstitial Deletion of Retinoblastoma (RB1) and Neurobeachin (NBEA) Is a Recurring Event in Multiple Myeloma.

Monoallelic chromosome 13 deletion detected by cytogenetics predicts poor patient survival in multiple myeloma (MM), but the genes responsible have not been conclusively identified. To this end, we performed array comparative genomic hybridization (aCGH) using a novel, human chromosome 13 oligonucleotide array (Nimblegen) with 385,272 probes and median probe spacing of 60 base pairs. The dense coverage, and the use of germline DNA collected from each patient as internal controls for DNA copy number polymorphisms, enabled unprecedented map resolution of somatic DNA gains and losses on chromosome 13. Array CGH was performed on genomic DNA isolated from CD138+ bone marrow plasma cells purified from 20 patients with MM, monoclonal gammopathy of undetermined significance (MGUS), or amyloidosis. Visual analysis of the aCGH data identified 4 patients with chromosome 13 interstitial deletions that were confirmed using a circular binary segment algorithm (Nimblegen). Monosomy chromosome 13 was detected in 5 patients by cytogenetics, and as expected, appeared normal by aCGH due to data normalization. We also performed an unsupervised analysis of the data, which identified 49 genes with DNA copy number decreases. Both methods identified copy number decreases at 13q14 commonly affected in MM and MGUS patients and thought to harbor a relevant tumor suppressor. Three of the 4 patients with interstitial deletions at 13q14 had striking regions of DNA copy loss whose minimally deleted region was defined by a patient with a small deletion spanning exon 20 of RB1, encoding part of the functionally important ‘pocket domain' responsible for binding E2F transcription factors. We found RB1 protein levels in MM cell lines correlated with RB1 genomic copy number, and therefore considered the model that RB1 haploinsufficiency contributes to MM. However, we found Rb1 heterozygous (HET) and wild type (WT) mice had indistinguishable steady-state B, T and myeloid compartments in addition to plasma cell induction in response to sheep red blood cell stimulation. Disease burden was similar in HET vs. WT Rb1 mice in a model of NRAS induced tumorigenesis. These results suggest other genomic events cooperate with RB1 copy number loss in MM. Unexpectedly, we found the 3 patients that had an interstitial deletion of RB1 at 13q14 concomitantly harbored a separate interstitial loss at 13q13. Every patient with DNA copy number loss of RB1 also had DNA copy loss within 13q13 (5 patients who lost the entire chromosome and 3 patients with interstitial deletions). The minimally deleted region at 13q13 mapped to the 5′ end of Neurobeachin (NBEA), which encodes a Protein Kinase A (PKA) anchoring protein. We detected NBEA transcripts at low levels in normal human plasma cells. NBEA transcripts and protein were robustly expressed in 3/5 MM cell lines. This is the first report of coordinate copy number loss of RB1 and NBEA on chromosome 13 in MM. Taken together, our data suggest that chromosome 13 deletions in MM may target protein dose level of RB1 and at least one other gene, likely NBEA. Our data provide a novel rationale for future studies to examine the biological consequences of coordinate loss of NBEA and RB1.

Cell lineage-specific interactions between Men1 and Rb in neuroendocrine neoplasia

Inactivation of multiple endocrine neoplasia (MEN) type 1 gene (Men1) results in development of multiple endocrine tumors in Men1(+/-) mice and in humans. Intriguingly, loss of the wild-type retinoblastoma 1 (Rb) gene also leads to MEN-like phenotype in Rb(+/-) mice. To evaluate potential genetic interactions between these genes, we prepared and characterized Men1(+/-)Rb(+/-) compound mice in parallel with their parental genotypes. Men1 and Rb did not cooperate in tumor suppression, as demonstrated by comparable survival rates of Rb(+/-) and Men1(+/-)Rb(+/-) mice, absence of tumor growth acceleration and lack of novel neoplasms. Notably, the loss of the remaining copy of the wild-type Men1 and Rb was mutually exclusive in all tumors of Men1(+/-)Rb(+/-) mice, including pituitary anterior lobe and adrenal medulla neoplasms shared by Rb- and Men1-deficient phenotypes. Down-regulation of Men1 targets p18 and p27 and increased presence of phosphorylated-Rb were observed in Men1-deficient pheochromocytomas of Men1(+/-)Rb(+/-) and Men1(+/-) mice. At the same time, the RNA interference (RNAi) knock-down of Men1 mRNA resulted in increased apoptosis of Rb-deficient medullary thyroid carcinoma cells. These results demonstrate that, depending on cell lineage context, combined Men1 and Rb deficiency may be either redundant or detrimental to neoplastic growth. Identification of cell lineage-specific interactions between Men1 and Rb may have important implications for development of rationally designed therapeutic approaches.

BRCA1a has antitumor activity in TN breast, ovarian and prostate cancers

Breast cancer gene 1 (BRCA1) mutations predispose women to breast and ovarian cancers and men to increased risks for prostate cancer. We have previously showed BRCA1 splice variant BRCA1a/p110 to induce apoptosis of human breast cancer cells. In the current study, stable expression of BRCA1a/p110 resulted in inhibition of growth of estrogen receptor (ER)-positive and triple-negative (TN) human breast, ovarian, prostate and colon cancer cells and mouse fibroblast cells. Similar to wild-type BRCA1, only those cells with wild-type Rb were sensitive to BRCA1a-induced growth suppression and the status of p53 did not affect the ability of BRCA1a to suppress growth of tumor cells. BRCA1a also significantly inhibited tumor mass in nude mice bearing human CAL-51 TN breast cancer, ES-2 ovarian cancer and PC-3 prostate cancer xenografts. These results suggest that the majority of exon 11 sequences (residues 263-1365) are not required for the tumor suppressor function of BRCA1 proteins. This is the first report demonstrating antitumor activity of BRCA1a in human ER-positive and TN breast, hormone-independent ovarian and prostate cancer cells. Currently, there are no effective treatments against TN breast cancers and results from these studies will provide new treatments for one of the biggest needs in breast cancer research.