Opposing Effects of PI3K/Akt and Smad-Dependent Signaling Pathways in NAG-1-Induced Glioblastoma Cell Apoptosis

Nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) is a divergent member of the transforming growth factor-beta (TGF-β) superfamily. NAG-1 plays remarkable multifunctional roles in controlling diverse physiological and pathological processes including cancer. Like other TGF-β family members, NAG-1 can play dual roles during cancer development and progression by negatively or positively modulating cancer cell behaviors. In glioblastoma brain tumors, NAG-1 appears to act as a tumor suppressor gene; however, the precise underlying mechanisms have not been well elucidated. In the present study, we discovered that overexpression of NAG-1 induced apoptosis in U87 MG, U118 MG, U251 MG, and T98G cell lines via the intrinsic mitochondrial pathway, but not in A172 and LN-229 cell lines. NAG-1 could induce the phosphorylation of PI3K/Akt and Smad2/3 in all six tested glioblastoma cell lines, except Smad3 phosphorylation in A172 and LN-229 cell lines. In fact, Smad3 expression and its phosphorylation were almost undetectable in A172 and LN-229 cells. The PI3K inhibitors promoted NAG-1-induced glioblastoma cell apoptosis, while siRNAs to Smad2 and Smad3 decreased the apoptosis rate. NAG-1 also stimulated the direct interaction between Akt and Smad3 in glioblastoma cells. Elevating the level of Smad3 restored the sensitivity to NAG-1-induced apoptosis in A172 and LN-229 cells. In conclusion, our results suggest that PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced glioblastoma cell apoptosis.     

Transforming growth factor-beta signaling helps specify tumor type in DMBA and hormone-induced mammary cancers

To determine the role of transforming growth factor-beta (TGF-beta) signaling in mammary development and tumor formation, we previously generated transgenic mice that expressed a dominant-negative form of the TGF-beta type II receptor (DNIIR) under the control of DNA regulatory elements from the metallothionein promoter (MT-DNIIR-28). In this report, we tested the hypothesis that loss of TGF-beta signaling in the mammary gland alters the development of chemically or hormonally induced tumors in mice. Four groups of mice were used in the study: wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft, and wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft treated with the carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). Tumor-free survival over time, tumor growth rate, and tumor pathology were measured. Statistically significant differences in tumor free survival over time or tumor growth rate were not detected in wild-type versus transgenic mice treated with DMBA. In contrast, tumor-free survival was significantly altered in transgenic mice that were treated with the pituitary isograft alone with MT-DNIIR mice developing tumors more quickly. Alterations in the types of tumors that formed in wild-type versus MT-DNIIR DMBA-treated mice were detected. In wild-type mice, tumors with squamous differentiation or bicellular adenomyoepitheliomas were most common. Adenomyoepitheliomas were not detected in transgenic mice. Furthermore, there was reduced staining for alpha smooth muscle actin and keratin 14, markers for myoepithelial cells, in the glandular portion of tumors in transgenic mice. The pathology of tumors induced by pituitary isograft alone was also markedly different in wild-type and transgenic mice. All the tumors classified from wild-type mice demonstrated some form of squamous differentiation, whereas squamous differentiation was not detected in the pituitary-induced transgenic tumors. The results suggest that TGF-beta acts as a tumor suppressor for hormone-induced cancers and that TGF-beta has a role in determining tumor pathology by regulating myoepithelial or squamous differentiation, maintenance, or transformation.     

Alterations in components of the TGF-beta superfamily signaling pathways in human cancer

Signaling by transforming growth factor-beta (TGF-beta) superfamily ligands to the nucleus is mediated by type I and type II receptors and the intracellular signal transducers, the Smads. Alteration of some of the components of these pathways has been observed in human tumors. These alterations can be deletions or mutations, or downregulation of components that act positively in the pathway, or alternatively, amplification or overexpression of inhibitors of the pathways. The selection of these alterations during tumor progression and their correlation with clinical outcomes, such as survival, risk of recurrence after tumor resection or tendency for metastatic spread, suggest that many are involved in tumor progression. Here, we review the genetic alterations and epigenetic modifications that occur in different components of the TGF-beta superfamily signaling pathways in human tumors and we discuss their correlation with clinical outcome. The evidence suggests that not all alterations of the TGF-beta superfamily signaling pathway components in human cancer have an equivalent effect on tumor progression and we discuss what implications this has for our understanding of the role of TGF-beta signaling in human cancer.     

Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-beta inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-beta receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-beta receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-beta whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-beta induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-beta acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-beta regulates angiogenesis. Finally, TGF-beta suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-beta pathway. In conclusion, TGF-beta signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.     

Haploid loss of bax leads to accelerated mammary tumor development in C3(1)/SV40-TAg transgenic mice: reduction in protective apoptotic response at the preneoplastic stage.

The dramatic increase in apoptosis observed during the development of preneoplastic mammary lesions is associated with a significant elevation in Bax expression in C3(1)/SV40 large T antigen (TAg) transgenic mice. The significance of Bax expression during tumor progression in vivo was studied by generating double-transgenic mice carrying the C3(1)/TAg transgene and mutant alleles for bax. C3(1)/TAg transgenic mice carrying mutant bax alleles exhibited accelerated rates of tumor growth, increased tumor numbers, larger tumor mass and decreased survival rates compared with mice carrying wild-type bax. Accelerated tumorigenesis associated with the bax+/- genotype did not require the loss of function of the second bax allele. Thus, haploid insufficiency of bax is enough to accelerate tumor progression, suggesting that the protective effect of Bax is dose-dependent. While levels of apoptosis in the preneoplastic lesions, but not carcinomas, were reduced in bax+/- or bax-/- mice compared with bax+/+ mice, rates of cellular proliferation in mammary lesions were similar among all bax genotypes. These data demonstrate that bax is a critical suppressor of mammary tumor progression at the stage of preneoplastic mammary lesion development through the upregulation of apoptosis, but that this protective effect is lost during the transition from preneoplasia to invasive carcinoma.     

Overexpression of Smad2 reveals its concerted action with Smad4 in regulating TGF-beta-mediated epidermal homeostasis.

Members of the transforming growth factor-beta (TGF-beta) superfamily are critical regulators for epithelial growth and can alter the differentiation of keratinocytes. Transduction of TGF-beta signaling depends on the phosphorylation and activation of Smad proteins by heteromeric complexes of ligand-specific type I and II receptors. To understand the function of TGF-beta and activin-specific Smad, we generated transgenic mice that overexpress Smad2 in epidermis under the control of keratin 14 promoter. Overexpression of Smad2 increases endogenous Smad4 and TGF-beta 1 expression while heterozygous loss of Smad2 reduces their expression levels, suggesting a concerted action of Smad2 and -4 in regulating TGF-beta signaling during skin development. These transgenic mice have delayed hair growth, underdeveloped ears, and shorter tails. In their skin, there is severe thickening of the epidermis with disorganized epidermal architecture, indistinguishable basement membrane, and dermal fibrosis. These abnormal phenotypes are due to increased proliferation of the basal epidermal cells and abnormalities in the program of keratinocyte differentiation. The ectodermally derived enamel structure is also abnormal. Collectively, our study presents the first in vivo evidence that, by providing an auto-feedback in TGF-beta signaling, Smad2 plays a pivotal role in regulating TGF-beta-mediated epidermal homeostasis.     

Indole-3-carbinol prevents PTEN loss in cervical cancer in vivo

Indole-3-carbinol (I3C) is a phytochemical (derived from broccoli, cabbage, and other cruciferous vegetables) with proven anticancer efficacy including the reduction of cervical intraepithelial neoplasia (CIN) and its progression to cervical cancer. In a breast cancer cell line, I3C inhibited cell adhesion, spreading, and invasion associated with an upregulation of the tumor suppressor gene PTEN, suggesting that PTEN is important in inhibition of late stages in the development of cancer. The goal of this study was to determine the expression of PTEN during the development of cervical cancer and whether I3C affected expression of PTEN in vivo. We show diminished PTEN expression during the progression from low-grade to high-grade cervical dysplasia in humans and in a mouse model for cervical cancer, the K14HPV16 transgenic mice promoted with estrogen. The implication is that loss of PTEN function is required for this transition. Additionally, dietary I3C increased PTEN expression in the cervical epithelium of the transgenic mouse, an observation that suggests PTEN upregulation by I3C is one mechanism by which I3C inhibits development of cervical cancer.     

Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression

The role and characteristics of tumor-associated neutrophils (TAN) in cancer are poorly defined. We have recently shown that TAN can have anti-tumorigenic (N1) or pro-tumorigenic (N2) functions. An interesting unanswered question is how the phenotype of TAN is influenced by the ongoing evolvement of tumor microenvironment. We therefore studied the phenotype and effects of TAN at different time points during tumor progression. We used two models of murine tumor cancer cell lines—Lewis lung carcinoma (LLC) and AB12 (mesothelioma). Neutrophils were studied at early and late stages and compared to each other and to neutrophils from bone marrow/periphery of naïve mice. Although there was no difference in the number of neutrophils entering the tumor, we found that at early stages of tumor development, neutrophils were almost exclusively at the periphery of the tumor. Only at later stages, neutrophils were also found scattered among the tumor cells. We further found that TAN from early tumors are more cytotoxic toward tumor cells and produce higher levels of TNF-α, NO and H₂O₂. In established tumors, these functions are down-regulated and TAN acquire a more pro-tumorigenic phenotype. In line with this phenotype, only depletion of neutrophils at later stages of tumor development inhibited tumor growth, possibly due to their central location in the tumor. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the changes in TAN during time. Additional research on the functional role of TAN and differences between subsets of TAN is currently underway.     

TGF-beta and cancer

TGF-beta signaling regulates tumorigenesis and in human cancer its signaling pathways are often modified during tumor progression. Prior to initiation and early during progression TGF-beta acts upon the epithelium as a tumor suppressor, however at later stages it is often a tumor promoter. Over the years, many studies have focused on the epithelial cell autonomous role for TGF-beta, however, TGF-beta is not strictly limited to this compartment in vivo. Recent studies addressing TGF-beta mediated stromal-epithelial interactions have significantly improved our understanding related to the regulation of cancer. In addition, stromal fibroblast cell autonomous effects have been observed in response to TGF-beta stimulation. According to the current literature and experimental evidence, TGF-beta is a potent ligand that regulates carcinoma initiation, progression and metastasis through a broad and complex spectrum of interdependent interactions.     

Antiviral action of the tumor suppressor ARF

Oncogenic viruses frequently target the pathways controlled by tumor suppressor genes, suggesting an extra function for these proteins as antiviral factors. The control exerted by the tumor suppressor Arf on cellular proliferation is crucial to restrict tumor development; however, a potential contribution of Arf to prevent viral infectivity has remained unexplored. In the present study, we investigated the consequences of loss or increased expression of Arf on viral infection. Our results reveal that ARF expression is induced by interferon and after viral infection. Furthermore, we show that ARF protects against viral infection in a gene dosage-dependent manner, and that this antiviral action is mediated in part by PKR through a mechanism that involves ARF-induced release of PKR from nucleophosmin complexes. Finally, Arf-null mice were hypersensitive to viral infection compared to wild-type mice. Together, our results reveal a novel and unexpected role for the tumor suppressor ARF in viral infection surveillance.     

A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.

The simian virus 40 large T antigen induces tumors in a wide variety of tissues in transgenic mice, the precise tissues depending on the tissue specificity of the upstream region controlling T-antigen expression. Expression of mutant T antigens that contain a subset of the protein's activities restricts the spectrum of tumors induced. Others showed previously that expression of a mutant large T antigen containing the N-terminal 121 amino acids (T1-121) under control of the lymphotropic papovavirus promoter resulted in slow-growing choroid plexus tumors, whereas full-length T antigen under the same promoter induced rapidly growing CPR tumors, T-cell lymphomas, and B-cell lymphomas. In those instances, the alteration in tumor induction or progression correlated with inability of the mutant large T antigen to bind the tumor suppressor p53. In the study reported here, we investigated the capacity of an N-terminal T antigen segment (T1-127) expressed in conjunction with small t antigen under control of the rat elastase-1 (E1) promoter to induce pancreatic tumors. The results show that pancreases of transgenic mice expressing T1-127 and small t antigen display acinar cell dysplasia at birth that progresses to neoplasia. The average age to death in these mice is within the range reported for transgenic mice expressing full-length T antigen under control of the E1 promoter. These results indicate that sequestering p53 by binding is not required for the development of rapidly growing acinar cell carcinomas. In addition, we provide evidence that small t antigen is unlikely to be required. Finally, we show that the p53 protein in acinar cell carcinomas is wild type in conformation.     

Autophagy Signaling and the Cogwheels of Cancer

The downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival, and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haplo-insufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.     

Increased malignancy of Neu-induced mammary tumors overexpressing active transforming growth factor beta1

To determine if Neu is dominant over transforming growth factor beta (TGF-beta), we crossed mouse mammary tumor virus (MMTV)-Neu mice with MMTV-TGF-beta1(S223/225) mice expressing active TGF-beta1 in the mammary gland. Bigenic (NT) and Neu-induced mammary tumors developed with a similar latency. The bigenic tumors and their metastases were less proliferative than those occurring in MMTV-Neu mice. However, NT tumors exhibited less apoptosis and were more locally invasive and of higher histological grade. NT mice exhibited more circulating tumor cells and lung metastases than Neu mice, while NT tumors contained higher levels of phosphorylated (active) Smad2, Akt, mitogen-activated protein kinase (MAPK), and p38, as well as vimentin content and Rac1 activity in situ than tumors expressing Neu alone. Ex vivo, NT cells exhibited higher levels of P-Akt and P-MAPK than Neu cells. These were inhibited by the TGF-beta inhibitor-soluble TGF-beta type II receptor (TbetaRII:Fc), suggesting they were activated by autocrine TGF-beta. TGF-beta stimulated migration of Neu cells into surrounding matrix, while the soluble TGF-beta inhibitor abrogated motility and invasiveness of NT cells. These data suggest that (i) the antimitogenic and prometastatic effects of TGF-beta can exist simultaneously and (ii) Neu does not abrogate TGF-beta-mediated antiproliferative action but can synergize with TGF-beta in accelerating metastatic tumor progression.