The transcription factor Dmp1 is a Ras/HER2-activated haplo-insufficient tumor suppressor that activates the Arf/p53 pathway of cell-cycle arrest. Recent evidence suggests that Dmp1 may activate p53 independently of Arf in certain cell types. Here, we report findings supporting this concept with the definition of an Arf-independent function for Dmp1 in tumor suppression. We found that Dmp1 and p53 can interact directly in mammalian cells via the carboxyl-terminus of p53 and the DNA-binding domain of Dmp1. Expression of Dmp1 antagonized ubiquitination of p53 by Mdm2 and promoted nuclear localization of p53. Dmp1-p53 binding significantly increased the level of p53, independent of the DNA-binding activity of Dmp1. Mechanistically, p53 target genes were activated synergistically by the coexpression of Dmp1 and p53 in p53(-/-);Arf(-/-) cells, and genotoxic responses of these genes were hampered more dramatically in Dmp1(-/-) and p53(-/-) cells than in Arf(-/-) cells. Together, our findings identify a robust new mechanism of p53 activation mediated by direct physical interaction between Dmp1 and p53.

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.

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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.

The transcription factor Dmp1 is a Ras/HER2-activated haplo-insufficient tumor suppressor that activates the Arf/p53 pathway of cell-cycle arrest. Recent evidence suggests that Dmp1may activate p53 independently of Arf in certain cell types. Here, we report findings supporting this concept with the definition of an Arf-independent function for Dmp1 in tumor suppression. We found that Dmp1 and p53 can interact directly in mammalian cells via the carboxyl-terminus of p53 and the DNA-binding domain of Dmp1. Expression of Dmp1 antagonized ubiquitination of p53 by Mdm2 and promoted nuclear localization of p53. Dmp1–p53 binding significantly increased the level of p53, independent of the DNA-binding activity of Dmp1. Mechanistically, p53 target genes were activated synergistically by the coexpression of Dmp1 and p53 in p53 / ;Arf / cells, and genotoxic responses of these genes were hampered more dramatically in Dmp1 / and p53 / cells than in Arf / cells. Together, our findings identify a robust newmechanism of p53 activationmediated by direct physical interaction between Dmp1 and p53. Cancer Res; 72(7); 1–11. 2012 AACR.

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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

Dmp1 (cyclin D-interacting myb-like protein 1; Dmtf1) is a transcription factor isolated in yeast two-hybrid screen as a cyclin D2 binding partner. As a transcription factor, Dmp1 binds to nonameric DNA consensus sequences CCCG(G/T)ATG(T/C) to act as an activator or a repressor. Extensive molecular and genetic evidence links tumor suppressor function of Dmp1 to regulation of the ARF-p53 pathway. In fact, Dmp1 directly binds to p19Arf (p14ARF in humans) promoter and induces its expression. Increase in ARF level leads to protection of p53 from Mdm2-mediated degradation and subsequent increase in p53 target genes involved in cell cycle arrest and apoptosis. Oncogenic Ras and Her2/neu induce Dmp1 expression leading to Arf-, p53-dependent cell cycle arrest. Human DMP1 locus is hemizygously deleted in ∼50% of breast carcinomas, which is mutually exclusive of ARF and p53 loss. Based on the amino acid sequence, predicted molecular weight of Dmp1 is 85kDa. However, Dmp1 migrates to ∼120-130kDa on a SDS-PAGE gel, suggesting post-translational modifications. Although signal transduction pathways regulating Dmp1 transcripton have been studied, the role of Dmp1 protein modification is unknown. Treatment of Dmp1 protein with calf intestine phosphatase and running a 2D SDS-PAGE gel, we have shown that one mode of post-translational modification is via phosphorylation. The presence of phosphates on several Serines and/or Threonines was confirmed with mass spectrometry. Using an Arf luciferase reporter assay in 3T3 cells, we found that MEKK1 (MEK kinase 1) synergizes with Dmp1 on the Arf promoter activity, with the synergy attenuated when several of the putative phosphorylation sites on Dmp1 were mutated. The effect observed was Dmp1-dependent since MEKK1 was unable to activate the Arf promoter in Dmp1-null cells. Full length (inactive) MEKK1 was unable to synergize with Dmp1 on the Arf promoter activity suggesting necessity of the kinase activity. The ability of MEKK1 to directly phosphorylate Dmp1 was confirmed in in vitro kinase assay using purified MEKK1 and Flag-Dmp1 proteins and Arf reporter assays where known kinases downstream of MEKK1(MEK1, MEK4/7, JNK1/2) were inhibited or knocked down. MEKK1 and Dmp1 co-immunoprecipitated when expressed in 3T3 cells. To study the effect of MEKK1 on endogenous Dmp1 and Arf, we either expressed MEKK1 or treated cells with MEKK1-activating drugs, cisplatin and etoposide. In this setting, endogenous p14ARF was induced and the banding pattern of Dmp1 protein was shifted in a SDS-PAGE gel indicating protein modification. Here we show that Dmp1 can be directly phosphorylated by MEKK1 to modulate Dmp1 transcriptional activity on the Arf promoter. Therefore, MEKK1 may activate the Arf-p53 pathway via direct Dmp1 protein phosphorylation. 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 3076. doi:10.1158/1538-7445.AM2011-3076

Cancer is caused by multiple genetic alterations leading to uncontrolled cell proliferation through multiple pathways. Malignant cells arise from a variety of genetic factors, such as mutations in tumor suppressor genes (TSGs) that are involved in regulating the cell cycle, apoptosis, or cell differentiation, or maintenance of genomic integrity. Tumor suppressor mouse models are the most frequently used animal models in cancer research. The anti-tumorigenic functions of TSGs, and their role in development and differentiation, and inhibition of oncogenes are discussed. In this review, we summarize some of the important transgenic and knockout mouse models for TSGs, including Rb, p53, Ink4a/Arf, Brca1/2, and their related genes.

Cancer is caused by multiple genetic alterations leading to uncontrolled cell proliferation through multiple pathways. Malignant cells arise from a variety of genetic factors, such as mutations in tumor suppressor genes (TSGs) that are involved in regulating the cell cycle, apoptosis, or cell differentiation, or maintenance of genomic integrity. Tumor suppressor mouse models are the most frequently used animal models in cancer research. The anti-tumorigenic functions of TSGs, and their role in development and differentiation, and inhibition of oncogenes are discussed. In this review, we summarize some of the important transgenic and knockout mouse models for TSGs, including Rb, p53, Ink4a/Arf, Brca1/2, and their related genes.

Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis.

Cyclin D binding myb-like protein 1 (Dmp1; Dmtf1) is a tumor suppressor that is deleted in ∼40 % of human non-small cell lung carcinomas. The Dmp1 promoter receives signals from oncogenic Ras and the protein shows its activity as a tumor suppressor by directly binding and transactivating the Arf promoter, and thereby inducing Arf-, p53-dependent cell cycle arrest. Both Eµ-Myc and K-ras LA -induced tumor development was accelerated in Dmp1 +/− and Dmp1 −/− mice, suggesting that Dmp1 is haplo-insufficient for tumor suppression. In Eµ-Myc lymphomas, the combined frequencies of p53 mutation and Arf deletion in the Dmp1 +/− or Dmp1 −/− mice were significantly lower (∼10 %) than that in Dmp1 +/+ littermates (∼50 %), indicating that Dmp1 is a physiological regulator of the Arf-p53 pathway in vivo. We recently found that the frequency of p53 mutation (∼40 %) was significantly decreased in both Dmp1 +/− and Dmp1 −/− backgrounds ( LA lung cancer model where the Ink4a/Arf involvement is very rare. Moreover, our recent data show that Dmp1 physically interacts with p53 to neutralize the activity of Hdm2. To demonstrate the Arf-independent function of Dmp1 on p53 activation in vivo, we injected doxorubicin into wild-type, Arf-null, and Dmp1-null mice, harvested the thymus at 2 hr intervals, and studied the expression of the p53 targets, p21 cip1 and bbc3, by real-time PCR and immunohistochemistry. Thymus was chosen as a target tissue since Dmp1 is highly expressed in the medulla in our histochemical studies. Western blotting analyses with DO-1 and phosphoserine-specific antibodies verified p53 activation by doxorubicin. Cleaved caspase 3 was detectable in the thymic medulla of wild-type mice 6 hrs after tail injection of doxorubicin, indicating the induction of apoptotic cell death by the genotoxic drug. Cleaved caspase 3 staining was significantly decreased in the thymus from Arf −/− mice as compared to wild-type mice, but the staining was even lower in Dmp1 −/− mice. Consistent with these findings, the p21 cip1 mRNA induction (∼23-fold increase in wild-type mice) was more significantly compromised in Dmp1 −/− mice (∼7-fold) than in Arf −/− mice (∼15-fold). Bbc was undetectable in Dmp1 −/− thymus even 4-6 hrs after injection of doxorubicin while it was significantly induced (∼3-fold) in both wild-type and Arf −/− mice. These mRNA data were confirmed by immunohistochemical staining of thymic tissues with p21 cip1 and bbc3-specific antibodies. Together, our data demonstrate the Arf-independent activation of p53 by Dmp1 in response to genotoxic stress in vivo. We are currently conducting the same series of experiments in the mouse lung. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1096.

The use of biomarkers ensures breast cancer patients receive optimal treatment. Established biomarkers such as estrogen receptor (ER) and progesterone receptor (PR) have been playing significant roles in the selection and management of patients for endocrine therapy. HER2 is a strong predictor of response to trastuzumab. Recently, the roles of ER as a negative and HER2 as a positive indicator for chemotherapy have been established. Ki67 has traditionally been recognized as a poor prognostic factor, but recent studies suggest that measurement of Ki67-positive cells during treatment will more effectively predict treatment efficacy for both anti-hormonal and chemotherapy. p53 mutations are found in 20–35% of human breast cancers and are associated with aggressive disease with poor clinical outcome when the DNA-binding domain is mutated. The utility of cyclin D1 as a predictor of breast cancer prognosis is controversial, but cyclin D1b overexpression is associated with poor prognosis. Likewise, overexpression of the low molecular weight form of cyclin E1 protein predicts poor prognosis. Breast cancers from BRCA1/2 carriers often show high nuclear grades, negativity to ER/PR/HER2, and p53 mutations, and thus, are associated with poor prognosis. The prognostic values of other molecular markers, such as p14ARF, TBX2/3, VEGF in breast cancer are also discussed. Careful evaluation of these biomarkers with current treatment modality is required to determine whether their measurement or monitoring offer significant clinical benefits.

Download an epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated hile it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is wn. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3′-kinaseF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpresoth Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu and mammary tumorigenesis was significantly accelerated in both Dmp1 and Dmp1 mice. Seleceletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu omas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1, Dmp1, and pe neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21, ng p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, enous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study endog shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis. Cancer Res; 70(22); 9084–94. ©2010 AACR.

Dmp1 (Dmtf1) encodes a Myb‐like transcription factor implicated in tumor suppression through direct activation of the Arf‐p53 pathway. The human DMP1 gene is frequently deleted in non‐small cell lung cancers, especially those that retain wild‐type INK4a/ARF and/or p53. To identify novel genes that are regulated by Dmp1, transcriptional profiles of lung tissue from Dmp1‐null and wild‐type mice were generated using the GeneChip Microarray. Comparative analysis of gene expression changes between the two groups resulted in identification of numerous genes that may be regulated by Dmp1. Notably, amphiregulin (Areg), thrombospondin‐1 (Tsp‐1), JunB, Egr1, adrenomedullin (Adm), Bcl‐3 and methyl‐CpG binding domain protein 1 (Mbd1) were downregulated in the lungs from Dmp1‐null mice while Gas1 and Ect2 genes were upregulated. These target genes were chosen for further analyses since they are involved in cell proliferation, transcription, angiogenesis/metastasis, apoptosis, or DNA methylation, and thus could account for the tumor suppressor phenotype of Dmp1. Dmp1 directly bound to the genomic loci of Areg, Tsp‐1, JunB and Egr1. Significant upregulation or downregulation of the novel Dmp1 target genes was observed upon transient expression of Dmp1 in alveolar epithelial cells, an effect which was nullified by the inhibition of de novo mRNA synthesis. Interestingly, these genes and their protein products were significantly downregulated or upregulated in the lungs from Dmp1‐heterozygous mice as well. Identification of novel Dmp1 target genes not only provides insights into the effects of Dmp1 on global gene expression, but also sheds light on the mechanism of haploid insufficiency of Dmp1 in tumor suppression.

Mouse mammary tumor virus (MMTV) long terminal repeat (LTR)-driven transgenic mice are excellent models for breast cancer as they allow for the targeted expression of various oncogenes and growth factors in neoplastic transformation of mammary glands. Numerous MMTV-LTR-driven transgenic mouse models of breast cancer have been created in the past three decades, including MMTV-neu/ErbB2, cyclin D1, cyclin E, Ras, Myc, int-1 and c-rel. These transgenic mice develop mammary tumors with different latency, histology and invasiveness, reflecting the oncogenic pathways activated by the transgene. Recently, homologous sequences of the env gene of MMTV have been identified in approximately 40% of human breast cancers, but not in normal breast or other types of cancers, suggesting possible involvement of mammary tumor virus in human breast carcinogenesis. Accumulating evidence demonstrates the association of MMTV provirus with progesterone receptor, p53 mutations and advanced-stage breast cancer. Thus, the detection of MMTV-like sequences may have diagnostic value to predict the clinical outcome of breast cancer patients.