Heterozygosity of p21WAF1/CIP1 enhances tumor cell proliferation and cyclin D1-associated kinase activity in a murine mammary cancer model.

The p21(WAF1/cIP1) cyclin-dependent kinase (cdk) inhibitor is a regulator of the G(1)-S cell cycle checkpoint. Despite the importance of p21 in cell cycle inhibition, its role as a tumor suppressor is uncertain. p21 mutations are infrequent in human tumors, and p21 null mice exhibit no increased tumor incidence. To ascertain whether p21 could influence tumor formation or progression in the context of other oncogenic stimuli, we crossed p21-deficient mice with mammary tumor susceptible Wnt-1 transgenic mice. The p21+/+, p21+/-, and p21-/- Wnt-1 transgenic female offspring were monitored for mammary tumor incidence and growth rates. p21 status had no effect on the age at which mammary tumors formed. However, p21+/- mammary tumors grew significantly faster than p21+/+ and p21-/- mammary tumors. The increased growth rates were confirmed by mitotic index counts and by BrdUrd labelling assays, indicating that a significantly higher percentage of p21+/- tumor cells were in S phase and mitosis than their p21+/+ and p21-/- counterparts. Moreover, cyclin D1-associated phosphorylation of retinoblastoma protein was significantly increased in p21+/- tumor lysates compared with p21+/+ and p21-/- lysates. These results are consistent with data indicating that reduced levels of p21 can facilitate cyclin/cdk complex formation while enhancing cdk activity. Thus, a reduction of p21 dosage may promote tumor progression in the presence of other oncogenic initiators. The dependence of p21 on prior oncogenic stimuli for its tumor-promoting activities suggests that it may behave as a tumor modifier gene rather than as a tumor suppressor gene.     

Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.     

p18 Ink4c and Pten constrain a positive regulatory loop between cell growth and cell cycle control

Inactivation of the Rb-mediated G1 control pathway is a common event found in many types of human tumors. To test how the Rb pathway interacts with other pathways in tumor suppression, we characterized mice with mutations in both the cyclin-dependent kinase (CDK) inhibitor p18 Ink4c and the lipid phosphatase Pten, which regulates cell growth. The double mutant mice develop a wider spectrum of tumors, including prostate cancer in the anterior and dorsolateral lobes, with nearly complete penetrance and at an accelerated rate. The remaining wild-type allele of Pten was lost at a high frequency in Pten+/- cells but not in p18+/- Pten+/- or p18-/- Pten+/- prostate tumor cells, nor in other Pten+/- tumor cells, suggesting a tissue- and genetic background-dependent haploinsufficiency of Pten in tumor suppression. p18 deletion, CDK4 overexpression, or oncoviral inactivation of Rb family proteins caused activation of Akt/PKB that was recessive to the reduction of PTEN activity. We suggest that p18 and Pten cooperate in tumor suppression by constraining a positive regulatory loop between cell growth and cell cycle control pathways.     

PRDX6 promotes tumor development via the JAK2/STAT3 pathway in a urethane-induced lung tumor model

Peroxiredoxin 6 (PRDX6) is a bifunctional protein with both glutathione peroxidase (GPx) and iPLA2 activities. Even though several pathophysiological functions have been studied, the definitive role of PRDX6 in tumor growth is not clear. Here, we compared carcinogen-induced tumor growth in PRDX6-transgenic (Tg) mice and non-Tg mice to evaluate the roles of PRDX6 in lung tumor development. Urethane (1 g/kg)-induced tumor incidence in PRDX6-Tg mice was significantly higher compared to non-Tg mice. In the tumors of PRDX6-Tg mice, the activation of JAK2/STAT3 and STAT3 DNA binding were also increased, accompanied by increased GPx and iPLA2 activities. PRDX6 was colocalized with JAK2 in tumor tissues and lung cancer cells and also showed physical interaction with JAK2. We found that increasing levels of PRDX6 increase the activation of the JAK2/STAT3 pathway. Furthermore, PRDX6-Tg mice showed altered cytokine levels in the tumors, especially leading to increased CCL5 levels. We validated that the activation of JAK2 was also decreased in lung tumors of CCR5^−/− mice, and CCL5 increased the JAK2/STAT3 pathway in the lung cancer cells. Thus, our findings suggest that PRDX6 promotes lung tumor development via its mediated and CCL5-associated activation of the JAK2/STAT3 pathway.     

HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization

The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice. LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.     

Inhibin and p27 interact to regulate gonadal tumorigenesis

Tumor suppressors function as antiproliferative signaling proteins, and defects in these genes lead to uncontrolled cell proliferation and cancer. For example, absence of the tumor suppressor p27(Kip1), a cyclin-dependent kinase inhibitor (CKI), results in increased body size, hyperplasia of several organs including the testes, and cancer in mice. Similarly, lack of inhibins, alpha/beta heterodimeric members of the transforming growth factor-beta (TGFbeta) superfamily, causes testicular and ovarian tumors of the granulosa/Sertoli cell lineage beginning at 4 weeks of age and adrenal tumors in gonadectomized mice. Neither the cell cycle alterations in the absence of inhibin nor the cause of the increased testis size in the p27 knockout mice is known. To study the molecular (cell cycle) changes that result from absence of inhibins, we analyzed the regulation of cell cycle proteins in gonadal tumors derived from inhibin alpha knockout mice (Inha(-/-)). Northern blot analyses demonstrate that cyclin-dependent kinase 4 (Cdk4) and cyclin D2 mRNA levels are elevated, and immunohistochemistry shows that p27 protein levels are decreased in both ovarian and testicular tumors from Inha(-/-) mice. These findings suggest that increased Cdk4/cyclin D2 (positive) activity and decreased p27 (negative) activity is causal for gonadal tumor formation. To test this hypothesis, we generated double mutant mice lacking both p27 and inhibin alpha to determine whether the tumor suppressors p27 and inhibin have additive suppressor activity in the gonads. Like Inha(-/-) mice, p27(-/-)Inha(-/-) mice demonstrate elevated serum activin levels, ovarian and testicular tumors, and a resultant lethal cachexia-like syndrome. However, whereas 95% of the Inha(-/-) female mice die by 18 weeks of age, 100% of the p27(-/-)Inha(-/-) female mice are dead by 8 weeks. Similarly, 95% of the Inha(-/-) single mutant males die by 13 weeks while 100% of the p27(-/-)Inha(-/-) male mice die by 10 weeks. Moreover, tumor foci in p27(-/-)Inha(-/-) mice can be observed as early as 2 weeks of age in males and as early as 4 weeks in females. These findings demonstrate that absence of both inhibin and p27 in mice causes earlier development of ovarian and testicular tumors and earlier death compared with absence of inhibin alone.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Pituitary hypoplasia in Pttg-/- mice is protective for Rb+/- pituitary tumorigenesis

Pituitary tumor transforming gene (Pttg) is induced in pituitary tumors and associated with increased tumor invasiveness. Pttg-null mice do not develop tumors, but exhibit pituitary hypoplasia, whereas mice heterozygous for the retinoblastoma (Rb) deletion develop pituitary tumors with high penetrance. Pttg-null mice were therefore cross-bred with Rb+/- mice to test the impact of pituitary hypoplasia on tumor development. Before tumor development, Rb+/-Pttg-/- mice have smaller pituitary glands with fewer cycling pituitary cells and exhibit induction of pituitary p21 levels. Pttg silencing in vitro with specific short hairpin interfering RNA in AtT20 mouse corticotrophs led to a marked induction of p21 mRNA and protein levels, decreased RB phosphorylation, and subsequent 24% decrease in S-phase cells. Eighty-six percent of Rb+/-Pttg+/+ mice develop pituitary adenomas by 13 months, in contrast to 30% of double-crossed Rb+/-Pttg-/- animals (P < 0.01). Pituitary hypoplasia, associated with suppressed cell proliferation, prevents the high penetrance of pituitary tumors in Rb+/- animals, and is therefore a protective determinant for pituitary tumorigenesis.     

Inactivation of heat shock factor Hsf4 induces cellular senescence and suppresses tumorigenesis in vivo

Studies suggest that Hsf4 expression correlates with its role in cell growth and differentiation. However, the role of Hsf4 in tumorigenesis in vivo remains unexplored. In this article, we provide evidence that absence of the Hsf4 gene suppresses evolution of spontaneous tumors arising in p53- or Arf-deficient mice. Furthermore, deletion of hsf4 alters the tumor spectrum by significantly inhibiting development of lymphomas that are normally observed in the majority of mice lacking p53 or Arf tumor suppressor genes. Using mouse embryo fibroblasts deficient in the hsf4 gene, we have found that these cells exhibit reduced proliferation that is associated with induction of senescence and senescence-associated β-galactosidase (SA-β-gal). Cellular senescence in hsf4-deficient cells is associated with the increased expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins. Consistent with the cellular senescence observed in vitro, specific normal tissues of hsf4(-/-) mice and tumors that arose in mice deficient in both hsf4 and p53 genes exhibit increased SA-β-gal activity and elevated levels of p27 compared with wild-type mice. These results suggest that hsf4 deletion-induced senescence is also present in vivo. Our results therefore indicate that Hsf4 is involved in modulation of cellular senescence, which can be exploited during cancer therapy.     

O6-3-[131I]iodobenzylguanine: improved synthesis and further evaluation of a potential agent for imaging of alkylguanine-DNA alkyltransferase

O(6)-Benzylguanine derivatives with suitable radionuclides attached to the benzyl ring are potentially useful in the noninvasive imaging of the DNA repair protein, alkylguanine-DNA alkyltransferase (AGT). Previously, O(6)-3-[(131)I]iodobenzylguanine ([(131)I]IBG) was prepared using a two-step approach; we now report its synthesis in a single step by the radioiododestannylation of O(6)-3-(trimethylstannyl)benzylguanine in 85-95% radiochemical yield. The in vitro specific uptake of [(131)I]IBG in DAOY human medulloblastoma cells, in TE-671 human rhabdomyosarcoma cells and a CHO cell line transfected to express AGT was linear (r(2) = 0.9-1.0) as a function of cell density. After intravenous injection of [(131)I]IBG in athymic mice bearing TE-671 xenografts, tumor uptake was 1.38 +/- 0.34% ID/g at 0.5 h and declined at 2 and 4 h. Preadministration of O(6)-(3-iodobenzyl)guanine (IBG) at 0.5 h increased uptake not only in tumor but also in several normal tissues. Notable exceptions were thyroid (p < 0.05), lung (p <0.05) and stomach. After intratumoral injection of [(131)I]IBG in the same xenograft model, the uptake in tumors that were depleted of AGT by BG treatment (165.8 +/- 27.5% ID/g) was about 60% of that in control mice (272.4 +/- 48.2% ID/g; p < 0.05).     

The role of CXCR2/CXCR2 ligand biological axis in renal cell carcinoma

Renal cell carcinoma (RCC) accounts for 3% of new cancer incidence and mortality in the United States. Studies in RCC have predominantly focused on VEGF in promoting tumor-associated angiogenesis. However, other angiogenic factors may contribute to the overall angiogenic milieu of RCC. We hypothesized that the CXCR2/CXCR2 ligand biological axis represents a mechanism by which RCC cells promote angiogenesis and facilitate tumor growth and metastasis. Therefore, we first examined tumor biopsies and plasma of patients with metastatic RCC for levels of CXCR2 ligands, and RCC tumor biopsies for the expression of CXCR2. The proangiogenic CXCR2 ligands CXCL1, CXCL3, CXCL5, and CXCL8, as well as VEGF were elevated in the plasma of these patients and found to be expressed within the tumors. CXCR2 was found to be expressed on endothelial cells within the tumors. To assess the role of ELR(+) CXC chemokines in RCC, we next used a model of syngeneic RCC (i.e., RENCA) in BALB/c mice. CXCR2 ligand and VEGF expression temporally increased in direct correlation with RENCA growth in CXCR2(+/+) mice. However, there was a marked reduction of RENCA tumor growth in CXCR2(-/-) mice, which correlated with decreased angiogenesis and increased tumor necrosis. Furthermore, in the absence of CXCR2, orthotopic RENCA tumors demonstrated a reduced potential to metastasize to the lungs of CXCR2(-/-) mice. These data support the notion that CXCR2/CXCR2 ligand biology is an important component of RCC tumor-associated angiogenesis and tumorigenesis.     

Expression of IL-17 mRNA in ovarian cancer

IL-17 is considered as a proinflammatory cytokine. We have demonstrated IL-17 is an angiogenic factor and promotes tumor growth in murine tumor models. In this report, we investigated the expression of IL-17 mRNA by RT-PCR and the relationship between IL-17 expression and microvascular density in ovarian cancer. IL-17 mRNA was expressed in 11 (64.7%) of 17 ovarian cancer. And the average number of blood vessels observed in IL-17 positive tumors (173.4 +/- 55.1/mm(2)) was significantly higher than that in negative tumors (107.7 +/- 57.8/mm(2)). These results indicated IL-17 is expressed in a considerable proportion of ovarian cancer and promotes tumor angiogenesis. There was no significant relationship between IL-17 expression and clinicopathologic parameters.     

Lung tumor growth-promoting function of peroxiredoxin 6

This study compared lung tumor growth in PRDX6-overexpressing transgenic (Tg) mice and normal mice. These mice expressed elevated levels of PRDX6 mRNA and protein in multiple tissues. In vivo, Tg mice displayed a greater increase in the growth of lung tumor compared with normal mice. Glutathione peroxidase and calcium-independent phospholipase 2 (iPLA2) activities in tumor tissues of Tg mice were much higher than in tumor tissues of normal mice. Higher tumor growth in PRDX6-overexpressing Tg mice was associated with an increase in activating protein-1 (AP-1) DNA-binding activity. Moreover, expression of proliferating cell nuclear antigen, Ki67, vascular endothelial growth factor, c-Jun, c-Fos, metalloproteinase-9, cyclin-dependent kinases, and cyclins was much higher in the tumor tissues of PRDX6-overexpressing Tg mice than in tumor tissues of normal mice. However, the expression of apoptotic regulatory proteins including caspase-3 and Bax was slightly less in the tumor tissues of normal mice. In tumor tissues of PRDX6-overexpressing Tg mice, activation of mitogen-activated protein kinases (MAPKs) was much higher than in normal mice. In cultured lung cancer cells, PRDX6 siRNA suppressed glutathione peroxidase and iPLA2 activities and cancer cell growth, but the enforced overexpression of PRDX6 increased cancer cell growth associated with their increased activities. In vitro, among the tested MAPK inhibitors, c-Jun NH₂-terminal kinase (JNK) inhibitor clearly suppressed the growth of lung cancer cells and AP-1 DNA binding, glutathione peroxidase activity, and iPLA2 activity in normal and PRDX6-overexpressing lung cancer cells. These data indicate that overexpression of PRDX6 promotes lung tumor growth via increased glutathione peroxidase and iPLA2 activities through the upregulation of the AP-1 and JNK pathways.     

Astrocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas.

Loss or mutation of p53 is thought to be an early event in the malignant transformation of many human astrocytic tumors. To better understand the role of p53 in their growth and transformation, we developed a model employing cultured neonatal astrocytes derived from mice deficient in one (p53 +/-) or both (p53 -/-) p53 alleles, comparing them with wild-type (p53 +/+) cells. Studies of in vitro and in vivo growth and transformation were performed, and flow cytometry and karyotyping were used to correlate changes in growth with genomic instability. Early-passage (EP) p53 -/- astrocytes achieved higher saturation densities and had more rapid growth than EP p53 +/- and +/+ cells. The EP p53 -/- cells were not transformed, as they were unable to grow in serum-free medium or in nude mice. With continued passaging, p53 -/- cells exhibited a multistep progression to a transformed phenotype. Late-passage p53 -/- cells achieved saturation densities 50 times higher than those of p53 +/+ cells and formed large, well-vascularized tumors in nude mice. p53 +/- astrocytes exhibited early loss of the remaining wild-type p53 allele and then evolved in a manner phenotypically similar to p53 -/- astrocytes. In marked contrast, astrocytes retaining both wild-type p53 alleles never exhibited a transformed phenotype and usually senesced after 7 to 10 passages. Dramatic alterations in ploidy and karyotype occurred and were restricted to cells deficient in wild-type p53 following repeated passaging. The results of these studies suggest that loss of wild-type p53 function promotes genomic instability, accelerated growth, and malignant transformation in astrocytes.