Mark A. Subler

A Farnesyltransferase Inhibitor Induces Tumor Regression in Transgenic Mice Harboring Multiple Oncogenic Mutations by Mediating Alterations in Both Cell Cycle Control and Apoptosis

ABSTRACT The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)–v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV–v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV–v-Ha-ras mice were interbred with p53−/− mice. Tumors inras/p53−/− mice treated with L-744,832 regressed as efficiently as MMTV–v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV–v-Ha-ras mice were also interbred with MMTV–c-myc mice to determine whetherras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV–v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/mycmice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV–c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

Role of LPA4/p2y9/GPR23 in negative regulation of cell motility.

Lysophosphatidic acid (LPA) is a ligand of multiple G protein-coupled receptors. The LPA(1-3) receptors are members of the endothelial cell differentiation gene (Edg) family. LPA(4)/p2y9/GPR23, a member of the purinergic receptor family, and recently identified LPA(5)/GPR92 and p2y5 are structurally distant from the canonical Edg LPA receptors. Here we report targeted disruption of lpa(4) in mice. Although LPA(4)-deficient mice displayed no apparent abnormalities, LPA(4)-deficient mouse embryonic fibroblasts (MEFs) were hypersensitive to LPA-induced cell migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase pathway by LPA(4), LPA(4) deficiency potentiated Akt and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA(4) converted LPA(4)-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA(4) strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA(1) in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA(4) attenuated LPA(1)-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA(4) is a suppressor of LPA-dependent cell migration and invasion in contrast to the motility-stimulating Edg LPA receptors.

Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease

Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62(P392L) mutation contributes to the development of this disease. To determine the effects of p62(P392L) expression on osteoclasts in vitro and in vivo, we introduced either the p62(P392L) or WT p62 gene into normal osteoclast precursors and targeted p62(P392L) expression to the osteoclast lineage in transgenic mice. p62(P392L)-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-kappaB ligand (RANKL) and TNF-alpha and showed increased NF-kappaB signaling but did not demonstrate increased 1,25-(OH)(2)D(3) responsivity, TAF(II)-17 expression, or nuclear number per osteoclast. Mice expressing p62(P392L) developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62(P392L) expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.

Expression of Measles Virus Nucleocapsid Protein in Osteoclasts Induces Paget's Disease‐Like Bone Lesions in Mice

We targeted the MVNP gene to the OCL lineage in transgenic mice. These mice developed abnormal OCLs and bone lesions similar to those found in Pagets patients. These results show that persistent expression of MVNP in OCLs can induce pagetic‐like bone lesions in vivo.

Induction of Apoptosis in BCR/ABL+ Cells By Histone Deacetylase Inhibitors Involves Reciprocal Effects on the RAF/MEK/ERK and JNK Pathways

Signal transduction events regulating induction of apoptosis by the histone deacetylase inhibitors (HDIs) sodium butyrate (SB) and SAHA have been examined in Bcr/Abl+ human leukemia cells (K562, LAMA 84). Exposure of K562 cells to >= 3.0 mM SB or 3.0 mM SAHA for 24-48 hr resulted in a marked induction of mitchondrial damage (e.g., cytochrome c release) and apoptosis, events associated with down-regulation of Bcr/Abl and Raf-1, induction of p21CIP1, inactivation of MEK1/2, ERK1/2, and p70S6K, and a dramatic increase in JNK activation. HDI-mediated apoptosis was attenuated by pharmacologic JNK inhibitors and enhanced by the MEK1/2 inhibitor U0126, which increased JNK activation, as well as by the JNK activator anisomycin. Furthermore, HDI lethality was significantly diminished in cells ectopically expressing constitutively active MEK1/2, confirming a functional role for MEK/ERK inactivation in HDI-mediated apoptosis. Similar events were observed in Bcr/Abl+ LAMA 84 cells. Lastly, the free radical scavenger L-N-acetylcysteine (LNAC) attenuated HDI-mediated ROS generation, JNK activation, and apoptosis. Together, these findings support a model in which induction of apoptosis in Bcr/Abl+ cells by HDIs involves coordinate inactivation of the cytoprotective Raf/MEK/ERK pathway in conjunction with the ROS-dependent activation of JNK.

A SQSTM1/p62 mutation linked to Paget’s disease increases the osteoclastogenic potential of the bone microenvironment

Pagets disease of bone (PDB) is the second most common bone disease and is characterized by focal bone lesions which contain large numbers of abnormal osteoclasts (OCLs) and very active normal osteoblasts in a highly osteoclastogenic marrow microenvironment. The etiology of PDB is not well understood and both environmental and genetic causes have been implicated in its pathogenesis. Mutations in the SQSTM1/p62 gene have been identified in up to 30% of Pagets patients. To determine if p62 mutation is sufficient to induce PDB, we generated mice harboring a mutation causing a P-to-L (proline-to-leucine) substitution at residue 394 (the murine equivalent of human p62(P392L), the most common PDB-associated mutation). Bone marrow cultures from p62(P394L) mice formed increased numbers of OCLs in response to receptor activator of NF-kappaB ligand (RANKL), tumor necrosis factor alpha (TNF-alpha) or 1alpha,25-(OH)(2)D(3), similar to PDB patients. However, purified p62(P394L) OCL precursors depleted of stromal cells were no longer hyper-responsive to 1alpha,25-(OH)(2)D(3), suggesting effects of the p62(P394L) mutation on the marrow microenvironment in addition to direct effects on OCLs. Co-cultures of purified p62(P394L) stromal cells with either wild-type (WT) or p62(P394L) OCL precursors formed more OCLs than co-cultures containing WT stromal cells due to increased RANKL production by the mutant stromal cells. However, despite the enhanced osteoclastogenic potential of both OCL precursors and marrow stromal cells, the p62(P394L) mice had histologically normal bones. These results indicate that this PDB-associated p62 mutation is not sufficient to induce PDB and suggest that additional factors acting together with p62 mutation are necessary for the development of PDB in vivo.

Astrocyte elevated gene-1 promotes hepatocarcinogenesis: Novel insights from a mouse model†‡

Astrocyte elevated gene‐1 (AEG‐1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG‐1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte‐specific expression of AEG‐1 (Alb/AEG1) was developed. Treating Alb/AEG‐1, but not wild‐type (WT) mice, with N‐nitrosodiethylamine resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis, and fatty acid synthesis. Hepatocytes isolated from Alb/AEG‐1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of prosurvival signaling pathways. Alb/AEG‐1 hepatocytes also exhibited marked resistance toward senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media from Alb/AEG‐1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG‐1 facilitated the association of factor XII (FXII) messenger RNA with polysomes, resulting in increased translation. Short interfering RNA–mediated knockdown of FXII resulted in profound inhibition of AEG‐1‐induced angiogenesis. Conclusion: We uncovered novel aspects of AEG‐1 functions, including induction of steatosis, inhibition of senescence, and activation of the coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG‐1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC. (HEPATOLOGY 2012;56:1782–1791)

ADAM10 Overexpression Shifts Lympho- and Myelopoiesis by Dysregulating Site 2/Site 3 Cleavage Products of Notch

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b+Gr-1+ cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncoupling the highly regulated proteolysis of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage−Sca-1+c-Kit+ cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary ligand for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch receptor demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage−Sca-1+c-Kit+ cells, respectively. In contrast, blockade of γ-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination.

Lung-specific expression of human mutant p53-273H is associated with a high frequency of lung adenocarcinoma in transgenic mice.

To investigate the tumorigenic potential of mutant p53 when selectively expressed in lung tissue, a transgenic mouse model was developed in which a mutant form of p53 (p53-273H) was placed under the transcriptional control of the lung-specific human surfactant protein C (SP-C) promoter. Two founder mice were identified, and a line of SP-C/p53-273H transgenic mice was established from one of the founders. Human p53-273H protein was detected specifically in lung tissue from transgenic mice. Malignant tumors, which were histologically characterized as adenocarcinomas, were observed in transgenic mice, with the earliest onset documented at 4 months of age. To further evaluate incidence and onset of tumor formation, transgenic mice (n=113) were sacrificed at age intervals ranging from 4–15 months. At 13–15 months of age, transgenic mice were significantly more likely to have lung tumors at necropsy than age-matched non-transgenic littermates (9 out of 39 (23%) versus 2 out of 35 (5.7%), χ2 test, P=0.036). The SP-C/p53-273H transgenic mice described here thus represent a genetically defined model with which to study the role of p53 mutations in lung tumorigenesis, as well as the potential complementary contributions of other genetic alterations or environmental carcinogens to lung tumor development.

ADAM8 enhances osteoclast precursor fusion and osteoclast formation in vitro and in vivo

ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate‐resistant acid phosphatase (TRAP)–ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP‐ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone‐resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF‐κB, Erk, and Akt signaling compared with wild‐type (WT) littermates. This increased bone‐resorbing capacity per OCL was associated with increased levels of p‐Pyk2 and p‐Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF‐α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF‐α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease.