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Supplementary Methods, Figures 1-6 from Critical Roles of DMP1 in Human Epidermal Growth Factor Receptor 2/neu-Arf-p53 Signaling and Breast Cancer Development

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. Cancer Res; 70(22); 9084–94. ©2010 AACR.

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. Cancer Res; 72(7); 1740–50. ©2012 AACR.

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.

FGFR2 fusions, amplifications, and mutations are oncogenic drivers that occur across multiple tumor types. Clinical efficacy observed with pan-FGFR inhibitors has validated the driver status of FGFR2 in FGFR2 fusion-positive intrahepatic cholangiocarcinoma (ICC), however, FGFR1-mediated toxicities (hyperphosphatemia, tissue mineralization) and the emergence of on-target FGFR2 resistance mutations limit the efficacy of pan-FGFR inhibitors. To overcome these limitations, we designed RLY-4008, a potent and highly selective, FGFR2 inhibitor. Despite significant investment in traditional structure-based drug design, selective targeting of FGFR2 has not been achieved. We leveraged differences in conformational dynamics between FGFR2 and other FGFR isoforms observed through molecular dynamics simulations to enable the design of RLY-4008. RLY-4008 inhibits FGFR2 with low nanomolar potency and demonstrates > 200-fold selectivity over FGFR1, and > 80- and > 5000-fold selectivity over FGFR3 and FGFR4, respectively, in biochemical assays. Additionally, RLY-4008 demonstrates high kinome selectivity for FGFR2 against a panel of > 400 human kinases. RLY-4008 has strong activity against primary and acquired FGFR2 resistance mutations in cellular assays, and potent antiproliferative effects on FGFR2-altered human tumor cell lines. In vivo, RLY-4008 demonstrates dose-dependent FGFR2 inhibition and induces regression in multiple human xenograft tumor models, including FGFR2 fusion-positive ICC, gastric, and lung cancers, FGFR2-amplified gastric cancer, and FGFR2-mutant endometrial cancer. Strikingly, RLY-4008 induces regression in an FGFR2 fusion-positive ICC model harboring the FGFR2V564F gatekeeper mutation and an endometrial cancer model harboring the FGFR2N549K mutation, two mutations that drive clinical progression on current pan-FGFR inhibitors. In the FGFR2V564F model, pan-FGFR inhibitors are ineffective, even at maximally tolerated doses. Notably, treatment of these tumors with RLY-4008 induces rapid regression and restores body weight. In rat and dog toxicology studies, RLY-4008 is well tolerated and is not associated with hyperphosphatemia or tissue mineralization at exposures significantly above those required to induce regression in all models. In contrast to pan-FGFR inhibitors, RLY-4008 is highly selective for FGFR2 and demonstrates strong activity against FGFR2 resistance mutations, suggesting that RLY-4008 may have broader therapeutic potential via preventing and overcoming therapeutic resistance. Together, these data and the favorable pharmaceutical properties of RLY-4008 strongly support its clinical development in FGFR2-altered tumors. Citation Format: Jessica Casaletto, Dejan Maglic, B. Barry Toure, Alex Taylor, Heike Schoenherr, Brandi Hudson, Kamil Bruderek, Songping Zhao, Patrick O'Hearn, Nastaran Gerami-Moayed, Demetri Moustakas, Roberto Valverde, Lindsey Foster, Hakan Gunaydin, Pelin Ayaz, Dina Sharon, Donald Bergstrom, James Watters. RLY-4008, a novel precision therapy for FGFR2-driven cancers designed to potently and selectively inhibit FGFR2 and FGFR2 resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1455.

The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap‐null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC. Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK‐JUN signaling, a well‐established tumor‐driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug‐resistant BCC.

Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo-insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functionally undefined isoforms, DMP1β and DMP1γ. In this study, we show that the DMP1 locus is alternatively spliced in ∼30% of breast cancer cases with relatively decreased DMP1α and increased DMP1β expression. RNA-seq analyses of a publicly available database showed significantly increased DMP1β mRNA in 43-55% of human breast cancers, dependent on histological subtypes. Similarly, DMP1β protein was found to be overexpressed in ∼60% of tumours relative to their surrounding normal tissue. Importantly, alteration of DMP1 splicing and DMP1β overexpression were associated with poor clinical outcomes of the breast cancer patients, indicating that DMP1β may have a biological function. Indeed, DMP1β increased proliferation of non-tumourigenic mammary epithelial cells and knockdown of endogenous DMP1 inhibited breast cancer cell growth. To determine DMP1β's role in vivo, we established MMTV-DMP1β transgenic mouse lines. DMP1β overexpression was sufficient to induce mammary gland hyperplasia and multifocal tumour lesions in mice at 7-18 months of age. The tumours formed were adenosquamous carcinomas with evidence of transdifferentiation and keratinized deposits. Overall, we identify alternative splicing as a mechanism utilized by cancer cells to modulate the DMP1 locus through diminishing DMP1α tumour suppressor expression, while simultaneously up-regulating the tumour-promoting DMP1β isoform.

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. Cancer Res; 72(7); 1740–50. ©2012 AACR.

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. Cancer Res; 70(22); 9084–94. ©2010 AACR.

SUMMARY Hippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional “omic” data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era., Graphical Abstract, In Brief Wang et al. perform a comprehensive analysis of 19 Hippo core genes across 33 cancer types using multidimensional “omic” data from The Cancer Genome Atlas. They characterize Hippo pathway activity by a YAP/TAZ transcriptional target signature of 22 genes and highlight the importance of Hippo signaling in squamous cell cancers.

Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo-insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functionally undefined isoforms, DMP1β and DMP1γ. In this study, we show that the DMP1 locus is alternatively spliced in ∼30% of breast cancer cases with relatively decreased DMP1α and increased DMP1β expression. RNA-seq analyses of a publicly available database showed significantly increased DMP1β mRNA in 43–55% of human breast cancers, dependent on histological subtypes. Similarly, DMP1β protein was found to be overexpressed in ∼60% of tumours relative to their surrounding normal tissue. Importantly, alteration of DMP1 splicing and DMP1β overexpression were associated with poor clinical outcomes of the breast cancer patients, indicating that DMP1β may have a biological function. Indeed, DMP1β increased proliferation of non-tumourigenic mammary epithelial cells and knockdown of endogenous DMP1 inhibited breast cancer cell growth. To determine DMP1β's role in vivo, we established MMTV-DMP1β transgenic mouse lines. DMP1β overexpression was sufficient to induce mammary gland hyperplasia and multifocal tumour lesions in mice at 7–18 months of age. The tumours formed were adenosquamous carcinomas with evidence of transdifferentiation and keratinized deposits. Overall, we identify alternative splicing as a mechanism utilized by cancer cells to modulate the DMP1 locus through diminishing DMP1α tumour suppressor expression, while simultaneously up-regulating the tumour-promoting DMP1β isoform.

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.

Breast cancer is the most common malignancy in women. Selection of patients for personalized therapy based on a risk-benefit assesment has not been improved. Dmp1 is a tumor suppressor that activates the p14ARF-p53 pathway to prevent breast cancer development. Regulation of Dmp1 at a transcription level by oncogenes is well understood; however, the role of Dmp1 phosphorylation is unknown. We identify a serine/threonine kinase of the MAPK pathway, MEKK1, which directly phosphorylates Dmp1. MEKK1 phosphorylates Dmp1 on multiple Serine/Threonines residues. Dmp1 and MEKK1 synergize on the Arf transactivation and the p53 pathway induction. Expression of MEKK1 in breast cancer cell lines leads to accumulation of ARF, p53, and p53 target genes. Analysis of MEKK1 locus in human breast tumor samples reveled that MEKK1 is frequently deleted and low expression of MEKK1 may be a new prognostic indicator of patient outcome.

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.

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.

Despite recent advances in therapeutic approaches and early detection, breast cancer remains significant health care burden in many developing countries. In fact, it has been suggested that up to ~30% of breast cancer cases are overdiagnosed as a result of early detection mammography screening. This finding poses a hypothesis that better patient stratification using prognostic/predictive indicators, especially for the patients with early diagnosis, would provide significant improvement in clinical management and reduce health care cost. Our recent work identified Dmp1 as a critical tumor suppressor in breast cancer. Dmp1 is a transcription factor that induces cell cycle arrest and senescence by activating the p14ARF-p53 pathway. Overexpression of Her2/neu activates the Dmp1 promoter via PI3K-Akt-NFκB pathway leading to increase of p53 target genes. Loss of Dmp1 accelerates mammary tumor development in MMTV-neu mouse model without difference between Dmp1+/- and Dmp1-/- genotypes. Human DMP1 locus on 7q21 is hemizygously deleted in ~42% of breast carcinomas, which is mutually exclusive of INK4a/ARF or p53 inactivation. In the cases with hemizygous DMP1 deletion, the other DMP1 allele remains wild-type without mutation or promoter hypermethylation, which suggests that DMP1 is haploinsufficient tumor suppressor. The hDMP1 locus encodes three distinct transcripts via alternative splicing of pre-mRNA at Exon 10. The bona fide tumor suppressor protein is named DMP1α, while the two other transcripts without known biological function were named DMP1β and DMP1γ. The qPCR analysis of 46 matched breast cancer samples revealed that 30% of tumor samples have splicing alteration of DMP1 to increase DMP1β isoform. Importantly, the patients with high DMP1β/α ratio in tumor samples had shorter relapse-free survival compared to those patients without splicing alteration. Furthermore, DMP1β/α ratio was increased in MMTV-neu mouse mammary tumors. Immunohistochemistry of 50 breast tumor samples showed that DMP1β protein is overexpressed which was also associated with shorter relapse-free survival. Expression of DMP1β in non-tumorigenic breast epithelial cell line, MCF10A, significantly increased the rate of cell proliferation and size of the mammospheres in Matrigel©. Knockdown of the endogenous DMP1β inhibits proliferation of breast cancer cell lines. To ascertain role of DMP1β in development of breast cancer in vivo, we developed a MMTV-DMP1β mouse model. The mammary glands from MMTV-DMP1β female mice show dysplastic morphological changes in the epithelium with multifocal tumors at 18 months of age. The tumor tissue and surrounding mammary glands were hyperproliferative as they expressed high levels of Ki67 and Cyclin D1. Overall, we provide evidence that alternative splicing to increase DMP1β expression leads to proliferation of human cells and mouse mammary gland and offers poor prognosis for breast cancer patients. Citation Format: Dejan Maglic, Robert Kendig, Mark Cline, Guangchao Sui. DMP1β, an alternative splice isoform of tumor suppressor hDMP1 locus, has oncogenic properties in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A013.

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

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

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

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.

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-rasLA-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 (< 10 %) in the K-rasLA 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, p21cip1 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 p21cip1 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 p21cip1 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.