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Supplementary Figure 1: Etv1 is required for intramuscular and myenteric ICC development. Supplementary Figure 2: Etv1 ablation with tamoxifen decreases Etv1 and Kit expression in Kit^delta558/+;Etv1^flox/flox;Rosa26^CreERT2/CreERT2 mice. Supplementary Figure 3: Etv1 positively regulates Kit expression in murine stomach. Supplementary Figure 4: ETV1 regulates KIT expression in GIST48 cells. Supplementary Figure 5: MAP kinase pathway inhibition decreases ETV1 target gene expression. Supplementary Figure 6: Dual lineage inhibition inhibits tumor initiation in vivo.

Supplementary Table 2: Gene sets enriched among genes DOWNregulated by tamoxifen treatment (Etv1 ablation) in murine GIST-like tumors.

Supplementary Table 3: Gene sets enriched among genes UPregulated by tamoxifen treatment (Etv1 ablation) in murine GIST-like tumors.

Supplementary Table 1: Differentially expressed genes (p1.5) between oil and tamoxifen treated tumors to conditionally ablate Etv1.

There is accumulating evidence that continuous activation of the sympathetic nervous system due to psychosocial stress increases resistance to therapy and accelerates tumor growth via β2-adrenoreceptor signaling (ADRB2). However, the effector mechanisms appear to be specific to tumor type. Here we show that activation of ADRB2 by epinephrine, increased in response to immobilization stress, delays the loss of MCL1 apoptosis regulator (MCL1) protein expression induced by cytotoxic drugs in prostate cancer cells; and thus, increases resistance of prostate cancer xenografts to cytotoxic therapies. The effect of epinephrine on MCL1 protein depended on protein kinase A (PKA) activity, but was independent from androgen receptor expression. Furthermore, elevated blood epinephrine levels correlated positively with an increased MCL1 protein expression in human prostate biopsies. In summary, we demonstrate that stress triggers an androgen-independent antiapoptotic signaling via the ADRB2/PKA/MCL1 pathway in prostate cancer cells.Implications:Presented results justify clinical studies of ADRB2 blockers as therapeutics and of MCL1 protein expression as potential biomarker predicting efficacy of apoptosis-targeting drugs in prostate cancer.

As one of few viral-positive cancers, nasopharyngeal carcinoma (NPC) is extremely rare across the world but very frequent in several regions of the world, including Southern China (known as the Cantonese cancer). Even though several genomic studies have been conducted for NPC, their sample sizes are relatively small and systematic comparison with other cancer types has not been explored. In this study, we collected four-hundred-thirty-one samples from six previous studies and provided the first integrative analysis of NPC genomes. Combining several statistical methods for detecting driver genes, we identified 25 novel drivers for NPC, including ATG14 and NLRC5. Many of these novel drivers are enriched in several important pathways, such as autophagy and immunity. By comparing NPC with many other cancer types, we found NPC is a unique cancer type in which a high proportion of patients (45.2%) do not have any known driver mutations (termed as “missing driver events”) but have a preponderance of deletion events, including chromosome 3p deletion. Through signature analysis, we identified many known and novel signatures, including single-base signatures (n = 12), double-base signatures (n = 1), indel signatures (n = 9) and copy number signatures (n = 8). Many of these new signatures are involved in DNA repair and have unknown etiology and genome instability, implying an unprecedented dynamic mutational process possibly driven by complex interactions between viral and host genomes. By combining clinical, molecular and intra-tumor heterogeneity features, we constructed the first integrative survival model for NPC, providing a strong basis for patient prognosis and stratification. Taken together, we have performed one of the first integrative analyses of NPC genomes and brought unique genomic insights into tumorigenesis of a viral-driven cancer.

Recently, exploration of stabler and lead-free perovskite absorbers with better cost-effective processability and prominent light-harvesting capacity has attracted extensive attentions. Inorganic C...

Recently, extensive studies have widely fabricated and demonstrated that organic-inorganic hybrid perovskite-type solar cells, CH3NH3PbI3, have the superior optoelectronic properties. We explore the structural, electronic, optical properties and stability of tetragonal phase mixed halide perovskites CH3NH3Pb(I1-yXy)3 (X = Cl, Br; y = 0, 0.25, 0.5, 0.75), using first-principles calculations. The calculation of electronic structure reveals that the valance band maximum is governed by the halide p-orbitals and Pb 6s-orbitals while the conduction band minimum formed by the mixed contribution from the p orbitals of Pb atom and s orbitals of I atom. For the stability and optical properties of CH3NH3Pb(I1-yXy)3, the absorption coefficients show a blueshift of the absorption onsets but their stabilities show gradually increase with the increasing of X contents. Three different doping occupation phases of MAPb(I0.5X0.5)3 demonstrate that the impact of doping positions in linear optical property cannot be neglected but there is no change in stability. On the basis of all calculated results, we conclude that the best accouplement between absorption coefficiency and stability can be achieved at y = 0.25 in CH3NH3Pb(I1−yBry)3 perovskites.

Structural, electrical, and optical properties, as well as the thermal stability, of lead free mixed halide perovskites, Cs2InBr5-yXy·H2O (X = Cl, I; y = 0, 1, 2, 3, 4, 5) and Cs2InBr6-x·xH2O (x = 1, 2, 3, 4) were investigated using first principles calculation. The structural stability of all studied pervoskites has been estimated by the calculation of structural parameters, Goldschmidt tolerance factor, and the octahedral factor. The obtained band gap of I-doped Cs2InBr5-yIy·H2O systems are 2.049, 1.726, 1.581, 1.475 and 1.272 eV with y =1, 2, 3, 4, 5, lower than 2.391 eV of Cs2InBr5·H2O. The absorption spectra were also calculated to evaluate optical properties of Cs2InBr5-yXy·H2O, which presents an obvious red-shift for I-doped perovskites in the visible region, while a blue-shift for Cl-doped perovskites. The formation energies of Cs2InBr5-yXy·H2O were calculated and the obtained negative values determined their good thermodynamic stability, except Cs2InBrI4·H2O and Cs2InI5·H2O with the extremely small positive values. In addition, the influence of the amount of crystal water on the properties of Cs2InBr6-x·xH2O was also considered. The defect formation energies of Cs2InBr6-x·xH2O were calculated and the obtained negative values indicate their appropriate stability. In brief, Cs2InBr3I2·H2O and Cs2InBr4·2H2O are two optimal candidates among all the studied systems for the photovoltaic application. These results provide some ideas for understanding various properties of mixed halide perovskites.

Recently, an anthraquinone-supported thiourea group linking a 1-aza-18-crown-6 macrocycle L has been the subject of extensive attention due to the perfect affinity towards metal cations. This work systematically researched the effects of different metal cations ([Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text]) on the second-order nonlinear optical (NLO) properties of macrocycle L by density functional theory (DFT). DFT calculations revealed that the values of first hyperpolarizabilities ([Formula: see text] decrease significantly when alkaline earth metal cations ([Formula: see text] and [Formula: see text]) were injected into macrocycle L due to the smaller charge transfer (CT) transition and larger transition energy. Conversely, the variations of [Formula: see text] values in alkali metal cations ([Formula: see text] and [Formula: see text] and transition metal cations ([Formula: see text] and [Formula: see text]) derivatives are not obvious compared to the [Formula: see text] value of macrocycle L. Therefore, the NLO properties of macrocycle can be effectively regulated by alkaline earth metal cations. Furthermore, we found that the [Formula: see text] value of anion-controlled complex Na(L)(ClO4) is larger than that of L*Na+ complex because the anion [Formula: see text] improves the planarity of anthraquinone-supported thiourea group leading to the enhancement of the CT ability. In addition, the influence of frequency-dependent on the first hyperpolarizabilities is weak for the current systems. Hence, we look forward to the conception of this work will offer a fundamental guideline and reference for further research for novel NLO materials.

Compared with sandstones and carbonates, volcanic reservoirs are much more complex and heterogeneous due to the special eruption diagenesis mechanism, many types of rock lithology, various mineral compositions and a broad wide of pore sizes according to previous studies. Consequently, accurate characterization of volcanic reservoirs using the powerful nuclear magnetic resonance (NMR) logging requires a comprehensive laboratory NMR investigation of volcanic rock because currently used NMR interpreted methods were only developed for sedimentary reservoirs. To gain an in-depth understanding of NMR characteristics of volcanic reservoirs with different lithology, a total of 108 low-permeability volcanic reservoir rock plugs from three large volcanic gas reservoirs named Xushen, Changling and Dixi, respectively, were prepared to perform NMR measurements and other related tests including CT scans, thin section petrography, mercury injection and mineral compositions analysis. The selected plugs comprise 9 types of lithology representing the main producing formation lithology of the three reservoirs. Specially, centrifuge tests were conducted with the maximum centrifugal forces up to 500 psi to explore the suitable capillary pressure for T2 cutoff determination. Results indicate that, obviously different from sandstone and carbonate plugs, NMR porosity of volcanic plugs at fully brine-saturated state is strongly dependent on rock lithology. NMR porosities of trachyte, trachytic volcanic and granite porphyry are significantly less than the conventional ones measured by the Archimedes method, which means that, accurate identification of reservoir intervals lithology is a primary prerequisite before correct interpretations of NMR logging. Paramagnetic minerals mainly iron and manganese elements contained in volcanic reservoirs are the fundamental cause resulting in this abnormal phenomenon. The critical values of iron and manganese elements contents are approximately 2% and 0.06% by weight, respectively, above which the NMR porosity will be considerably less than the conventional one suggesting by inductively coupled plasma-atomic emission spectrometry (ICP-AES) tests on 14 representative plugs. Then, a new NMR porosity corrected formula was developed to improve interpreted quality of NMR logging. It was found that the suitable capillary pressure for determination of T2 cutoff of volcanic reservoirs is 400psi, 3 times larger than the commonly recommended standard (100psi) for sandstones. The calculated T2 cutoff ranges from 3 to 120ms and its average values is 50ms. T2 cutoff between different volcanic reservoirs and lithologies exhibits significant difference. The laboratory NMR results were used to interpret NMR logging of the Xushen reservoir of Daqing oilfield in eastern China and aided in detailed reservoirs evaluation. The outcome of beneficial intervals selection and high productivity well completion based on the NMR logging interpretation is very encouraging. This study indicated that a comprehensive laboratory NMR tests is very essential to successful application of NMR logging for complex reservoirs such as volcanic reservoirs.

Extensive studies have demonstrated that all-inorganic lead-free halide perovskite solar cells as promising photoelectric devices are receiving great attention. In this paper, we have investigated the structures, electronical and optical properties of perovskite Cs2Ti(Br1-xYx)6 (Y = Cl, I, x ​= ​0, 0.25, 0.5, 0.75, 1). When x ​= ​0.25, the system Cs2Ti(Br0.75Cl0.25)6 has the suitable band gap value. With the increase of Cl content, the absorption coefficients showing blue shift which lead to the optical performance slightly decreased. But the systems become more stable. For the substitution of I to Br, the band gap values are lower than the optimal one. As to optical calculations, we find that the systems show obvious red shift, which result in the optical performance enhanced. However, the systems become unstable and not easy to be prepared. All calculations reveal that these excellent properties make Cs2Ti(Br0.75Cl0.25)6 as an ideal candidate material for perovskite solar cells.

Malignant peripheral nerve sheath tumors (MPNSTs) represent a group of highly aggressive soft-tissue sarcomas that may occur sporadically, in association with neurofibromatosis type I (NF1 associated) or after radiotherapy. Using comprehensive genomic approaches, we identified loss-of-function somatic alterations of the Polycomb repressive complex 2 (PRC2) components (EED or SUZ12) in 92% of sporadic, 70% of NF1-associated and 90% of radiotherapy-associated MPNSTs. MPNSTs with PRC2 loss showed complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3) and aberrant transcriptional activation of multiple PRC2-repressed homeobox master regulators and their regulated developmental pathways. Introduction of the lost PRC2 component in a PRC2-deficient MPNST cell line restored H3K27me3 levels and decreased cell growth. Additionally, we identified frequent somatic alterations of CDKN2A (81% of all MPNSTs) and NF1 (72% of non-NF1-associated MPNSTs), both of which significantly co-occur with PRC2 alterations. The highly recurrent and specific inactivation of PRC2 components, NF1 and CDKN2A highlights their critical and potentially cooperative roles in MPNST pathogenesis.

Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1-cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1-induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1(+/+);MMTV-cyclin D1 and Dmp1(+/+);MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer.

Gastrointestinal stromal tumor (GIST), originating from the interstitial cells of Cajal (ICC), is characterized by frequent activating mutations of the KIT receptor tyrosine kinase. Despite the clinical success of imatinib, which targets KIT, most patients with advanced GIST develop resistance and eventually die of the disease. The ETS family transcription factor ETV1 is a master regulator of the ICC lineage. Using mouse models of Kit activation and Etv1 ablation, we demonstrate that ETV1 is required for GIST initiation and proliferation in vivo, validating it as a therapeutic target. We further uncover a positive feedback circuit where MAP kinase activation downstream of KIT stabilizes the ETV1 protein, and ETV1 positively regulates KIT expression. Combined targeting of ETV1 stability by imatinib and MEK162 resulted in increased growth suppression in vitro and complete tumor regression in vivo. The combination strategy to target ETV1 may provide an effective therapeutic strategy in GIST clinical management. Significance: ETV1 is a lineage-specific oncogenic transcription factor required for the growth and survival of GIST. We describe a novel strategy of targeting ETV1 protein stability by the combination of MEK and KIT inhibitors that synergistically suppress tumor growth. This strategy has the potential to change first-line therapy in GIST clinical management. Cancer Discov; 5(3); 304–15. ©2015 AACR. See related commentary by Duensing, p. 231 This article is highlighted in the In This Issue feature, p. 213

Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1α transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21(Cip1), markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1α prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1α transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression.

TBX3 is a transcriptional repressor belonging to the T-box gene family. It is involved in lineage specification in mammary gland, limb and heart during embryonic development. Mutations in TBX3 can cause ulnar-mammary syndrome, affecting limb, apocrine gland, tooth, hair and genital development. TBX3 is amplified/mutated in a variety of cancers, including melanoma, bladder, breast and prostate cancer. Recent findings suggest there is a strict correlation between estrogen receptor and TBX3 expression in mammary gland and TBX3 is mutated in 9% of invasive lobular breast cancer. However, little is known about the functions of TBX3 in hormone-dependent tumors. Here we screened several AR+ and AR- prostate cancer cell line and we found that the expression of TBX3 and AR is positively correlated. Moreover, in benign human prostate tissue, TBX3 localization is overlapped with that of AR. Knocking down TBX3 by siRNA resulted in decreased cell proliferation and cell cycle arrest in AR+ cell line Lncap and Vcap, but there was no effect on AR- cell line PC3 and DU14 upon TBX3 knock-down. TBX3 ChIP-seq in both Lncap and Vcap revealed co-binding of TBX3, ETS transcriptional factors and FOXA1 on AR targeted genes. Gene Set Enrichment Analysis (GSEA) of RNA-seq data from Lncap cells infected with lentivirus expressing siSCR or siTBX3 indicated that TBX3 suppresses AR and ETV1 signaling. In conclusion, our data suggests that TBX3 is essential for AR+ prostate cancer cell growth and may play a role in regulating androgen signaling. Citation Format: sinan zhu. Elucidate the functions of TBX3 in AR positive prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2001.

Our recent study showed critical roles of Dmp1 as a sensor of oncogenic Ras, HER2/neu signaling and activation of the Arf-p53 pathway. To elucidate the role of human DMP1 (hDMP1) in breast cancer, one hundred and ten pairs of human breast cancer specimen were studied for the alterations of the hDMP1-ARF-Hdm2-p53 pathway with follow up of clinical outcomes. Loss of heterozygosity (LOH) of the hDMP1 locus was found in 42% of human breast carcinomas, while that of INK4a/ARF and p53 were found in 20 and 34%, respectively. Hdm2 amplification was found in 13% of the same sample, which was found independently of LOH for hDMP1. Conversely, LOH for hDMP1 was found in mutually exclusive fashion with that of INK4a/ARF and p53, and was associated with low Ki67 index and diploid karyotype. Consistently, LOH for hDMP1 was associated with luminal A category and longer relapse-free survival, while that of p53 was associated with non-luminal A and shorter survival. Thus, loss of hDMP1 could define a new disease category associated with prognosis of breast cancer patients. Human breast epithelial cells/cancer cells with wild-type p53 were sensitive to growth inhibition by activated Dmp1:ER while those that delete p14(ARF) or p53, and/or Hdm2 amplification showed partial or nearly complete resistance, indicating that p53 is a critical target for hDMP1 to exhibit its biological activity.

Small cell lung cancer (SCLC) makes up almost 15% of all cases of lung cancer and occurs almost exclusively in individuals with a history of smoking (Blackhall & Faivre-Finn, 2011; Meyerson et al., 2004; Tamasi and Muller, 2011; Walenkamp et al., 2009). However, SCLCs differ significantly from NSCLCs in specific genetic alterations that occur. Moreover, smoking-damaged bronchial epithelia accompanying SCLCs appears to have undergone significantly more acquired genetic damage than is frequently found in NSCLCs. Two subtypes of SCLC exist: homogeneous small cell carcinoma and combined SCLC (mixture of any non-small cell type) (Meyerson et al., 2004; Tamasi and Muller, 2011). SCLC in its advanced stage has an aggressive clinical course and is commonly accompanied by paraneoplastic syndromes. Autocrine growth factors, such as neuroendocrine regulatory peptides (e.g. bombesin/gastrin-releasing peptide), are prominent in SCLC. SCLC is categorized as limited stage disease (LS) when confined to the ipsilateral hemithorax and within a single radiation port, while extensive stage disease (ES) includes metastatic disease outside the ipsilateral hemithorax (Blackhall & Faivre-Finn, 2011; Meyerson et al., 2004; Tamasi and Muller, 2011; Walenkamp et al., 2009). SCLC is sensitive to chemotherapy; response rates to front-line agents are often in the range of 60%, with approximately 10% of patients achieving a complete response, even in the setting of metastatic disease (Brambilla et al., 2009 Jemal et al., 2006). Despite this, the relapse rates are quite high and survival with currently available salvage therapy is quite modest. With current therapy, patients with LSSCLC have a median survival of 17 months and a 5-year overall survival rate of 12% , while patients with ES-SCLC have a median survival of 8.9 months, and a 5-year survival rate of approximately 2%. (Brambilla et al., 2009 Jemal et al., 2006; Tamasi and Muller, 2011). This article will review the molecular targeted agents, the genetic abnormalities, and therapeutic efficacy in SCLC.

Breast cancer remains a significant public health issue in the industrialized countries. Physicians depend on surgery, cytotoxic adjuvant chemotherapy, or radiation for treatment of breast cancer patients. Few novel therapies are approved for clinical use. Although existing therapies are effective in controling the disease in many patients, the cytotoxic side-effects make them limited. This is especially the case in the patients with early stage breast cancer whose prognosis with surgery alone is good and avoiding adjuvant chemotherapy would spare them toxic side effects. On the other hand, significant fraction of patients relapses and develops metastasis. Therefore, it is critical to identify markers for better breast cancer patient stratification based on their prognosis that would guide physicians in selection and aggressiveness of therapies. Recently, we linked transcription factor DMP1 (cyclin D binding protein 1, Dmtf1) as a critical tumor suppressor that blocks proliferative signals from ErbB2 and oncogenic Ras by activating p14Arf-p53 pathway. hDMP1 is hemizygously deleted in ∼50% of human breast tumors that retain wild-type ARF and p53. In this study we identified an upstream kinase, MEKK1, which cooperates with Dmp1 to activate Arf transcription. In constitutively active form (C-terminal kinase domain or CA-MEKK1), MEKK1 increased transcriptional activity of Dmp1 via direct phosphorylation, while the full length (regulatory and kinase domain) form behaved as a feedback inhibitor by increasing Dmp1 ubiquitination. Phosphorylation sites on DMP1 that increase its transcriptional activity were mapped to the C-terminal transactivation domain. Expression of CA-MEKK1 in breast cancer cell lines activated endogenous Arf-p53 pathway. hMEKK1 is located on human 5q11 chromosome, a locus frequently deleted in breast and lung cancer. Since MEKK1 appeared to be an activator of p53 tumor suppressor pathway, we wondered if MEKK1 is involved in human cancer. Using DNA from breast cancer patient tumors and matched normal tissue, we analyzed hMEKK1 gene deletion using specific loss of heterozygosity (LOH) primers. MEKK1 was found hemizygously deleted in ∼20% of patients. Immunohistochemistry confirmed reduction of MEKK1 protein intensity to LOH(+) compared to LOH (-) patients. In the independent cohort of breast cancer patients, we show that low MEKK1 mRNA expression is associated with adverse outcome. Here we show that constitutively active MEKK1 is a novel activator of Dmp1-Arf-p53 pathway via Dmp1 phosphorylation. The MEKK1 gene was frequently deleted in breast tumor tissue and its expression was correlated with disease outcome. Overall, MEKK1 is a potential prognostic/predictive indicator for breast cancer and could be used by physicians to better stratify patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4158. doi:1538-7445.AM2012-4158

Cyclin D1 is a crucial regulator in mammalian cell cycle that binds to and activates CDK4/6, which is an kinase that drives cells to enter S phase. It has an important oncogenic role in breast cancer because the CCND1 gene is amplified in ∼15% of cases and its protein expression is elevated in up to 50% of cases. Previous studies demonstrate that cyclin D1 overexpression can cause pRb hyper-phosphorylation and therefore induce cell hyperproliferation, which can result in the development of mammary adenocarcinoma. In addition, cyclin D1 is found to contribute to tumorigenesis by affecting the activity of transcriptional factors via physical interaction. One of the affected transcriptional factors is called Dmp1, which acts as a tumor suppressor when receiving oncogenic signals. It activates Arf by binding to its promoter region, therefore causing Arf, p53-dependent cell cycle arrest in normal cells. Our recent study in MMTV-neu mice showed that loss of Dmp1 significantly accelerates mammary carcinogenesis. Although both Dmp1 and cyclin D1 play vital roles in breast cancer prevention and development respectively, the biological functions and significance of Dmp1-cyclin D1 interaction remains to be explored. To identify the impact of their interaction on the activation of p16Ink4a/ p19Arf promoters induced by cyclin D1, MEFs were overexpressed cyclin D1 and we found the endogenous p16Ink4a and p19Arf transcripts were increased up to 7 fold. However, their transcription level was not affected in Dmp1-null MEFs. Moreover, cyclin D1 mutant α142-253 that cannot bind to Dmp1could not fully activate the p16Ink4a/p19Arf promoters, indicating the activation of p16Ink4a/p19Arf promoters by cyclin D1 is dependent on Dmp1, and loss of Dmp1-cyclin D1 interaction will attenuate this effect. To identify the potential regulation of p16Ink4a by Dmp1, we performed EMSA and luciferase reporter assay, and we found that Dmp1 activates the p16Ink4a promoter by directly binding to its promoter. Moreover, the p16Ink4a mRNA and protein level were increased upon Dmp1 overexpression. The above results suggest that Dmp1 is a physiological regulator of p16Ink4a. To explore the effect of Dmp1 on Cdk4 kinase activity, 293T cells were overexpressed cyclin D1 alone or together with Dmp1, and we found there was less cyclin D1 associated with CDK4 after CDK4 immunoprecipitation. As a consequence, pRb phosphorylation was decreased in CDK4 kinase assay. These results suggest that Dmp1 decreases CDK4 kinase activity by impairing cyclin D1-CDK4 interaction. Taken together, our data suggest that Dmp1 decreases pRb phosphorylation in two ways: one is to directly bind to and activate p16INK4a, and the other one is to block the CDK4 kinase activity by binding to cyclin D1. In addition, cyclin D1-Dmp1 interaction is indispensible in activation of p16Ink4a/p19Arf promoters induced by cyclin D1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2052. doi:1538-7445.AM2012-2052

Cyclin D1 is a member of cyclin protein family, which drives the cell cycle transition from G1 to S phase. The cyclin D1 protein is overexpressed in ∼50% of human breast cancers and is associated with short survival of patients when the gene is amplified. Cyclin D1 forms holoenzymes with cyclin-dependent protein kinase (Cdk) 4 and 6, and the Cyclin D-Cdk4/6 complex phosphorylates and inactivates the pRb retinoblastoma tumor suppressor, releasing E2F transcriptional factors from Rb inhibition. The pRb phosphorylation is blocked by p16INK4a, encoded by the CDKN2A locus, which inhibits the kinase activity of Cyclin D-Cdk4/6 complex. The CDKN2A locus also encodes another tumor suppressor protein named p14ARF (p19Arf in mouse), which stabilizes p53 by inhibiting MDM2-dependent degradation. Previous studies from our laboratory showed that the cyclin D1 collaborated with the Dmp1 (cyclin D binding myb-like protein 1) tumor suppressor to activate the Arf promoter. To further demonstrate the effect of cyclin D1 on p19Arf and p16Ink4a promoter activity, NIH 3T3 cells and MEF cells were transfected a construct expressing the luciferase reporter gene linked with the Arf promoter together with vector or cyclin D1. Using luciferase reporter assay, we found that the Arf promoter was activated upon cyclin D1 overexpression in both cell lines, and the activation was abrogated when cells were co-transfected with E2F-DB, which displaces endogenous E2Fs and acts in a dominant-negative manner. Consistent with reporter assays, the Arf mRNA was increased in MEFs overexpressing cyclin D1, compared to those infected with vector only. By performing chromatin immunoprecipitation in tumors of mammary gland from MMTV-neu transgenic mice that overexpress cyclin D1, we found that cyclin D1 can interact with the Arf promoter although it cannot directly bind to the Arf promoter region in electrophoretic mobility shift assay using recombinant cyclin D1 protein. To identify the effect of cyclin D1 on p16Ink4a promoter, we performed luciferase reporter assay in NIH 3T3 cells by co-transfecting Ink4a promoter together with vector or cyclin D1. We found that the Ink4a promoter activity was increased upon cyclin D1 overexpression, and the activation was not abrogated when cells were transfected with cyclin D1-K114E that does not bind to CDK4. Taken together, our data suggest that overexpression of cyclin D1 activates both p19Arf and p16Ink4a promoters to prevent neoplastic growth of incipient cancer cells, and the activation is mediated by indirect binding of cyclin D1 to the promoter regions. We are currently mapping the cyclin D1-responsive elements on the Arf and Ink4a promoters to identify transcription factors responsible for Arf/Ink4a induction by cyclin D1. 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 2958. doi:10.1158/1538-7445.AM2011-2958