GRIM-19 mutations fail to inhibit v-Src-induced oncogenesis.

The non-receptor tyrosine kinase Src is a major player in multiple physiological responses including growth, survival and differentiation. Overexpression and/or oncogenic mutation in the Src gene have been documented in human tumors. The v-Src protein is an oncogenic mutant of Src, which promotes cell survival, migration, invasion and division. GRIM-19 is an antioncogene isolated using a genome-wide knockdown screen. Genes associated with Retinoid-IFN-induced Mortality (GRIM)-19 binds to transcription factor STAT3 and ablates its pro-oncogenic effects while v-Src activates STAT3 to promote its oncogenic effects. However, we found that GRIM-19 inhibits the pro-oncogenic effects of v-Src independently of STAT3. Here, we report the identification of functionally inactivating GRIM-19 mutations in a set of head and neck cancer patients. While wild-type GRIM-19 strongly ablated v-Src-induced cell migration, cytoskeletal remodeling and tumor metastasis, the tumor-derived mutants (L(71)P, L(91)P and A(95)T) did not. These mutants were also incapable of inhibiting the drug resistance of v-Src-transformed cells. v-Src downregulated the expression of Pag1, a lipid raft-associated inhibitor of Src, which was restored by wild-type GRIM-19. The tumor-derived mutant GRIM-19 proteins failed to upregulate Pag1. These studies show a novel mechanism that deregulates Src activity in cancer cells.

Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC.

The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy.

Enhanced tumor suppression in vitro and in vivo by co-expression of survivin-specific siRNA and wild-type p53 protein.

The development of malignant prostate cancer involves multiple genetic alterations. For example, alterations in both survivin and p53 are reported to have crucial roles in prostate cancer progression. However, little is known regarding the interrelationships between p53 and survivin in prostate cancer. Our data demonstrate that the expression of survivin is inversely correlated with that of wtp53 protein (r(s)=0.548) in prostate cancer and in normal prostate tissues. We have developed a therapeutic strategy, in which two antitumor factors, small interfering RNA-survivin and p53 protein, are co-expressed from the same plasmid, and have examined their effects on the growth of PC3, an androgen-independent prostate cancer cell line. When p53 was expressed along with a survivin-specific short hairpin RNA (shRNA), tumor cell proliferation was significantly suppressed and apoptosis occurred. In addition, this combination also abrogated the expression of downstream target molecules such as cyclin-dependent kinase 4 and c-Myc, while enhancing the expression of GRIM19. These changes in gene expression occurred distinctly in the presence of survivin-shRNA/wtp53 compared with control or single treatment groups. Intratumoral injection of the co-expressed construct inhibited the growth and survival of tumor xenografts in a nude mouse model. These studies revealed evidence of an interaction between p53 and survivin proteins plus a complex signaling network operating downstream of the wtp53-survivin pathway that actively controls tumor cell proliferation, survival and apoptosis.

GRIM-19 inhibits v-Src-induced cell motility by interfering with cytoskeletal restructuring.

GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling, especially the formation of podosomes and. Here, we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling, that is, podosome formation. We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations.

GRIM-19 associates with the serine protease HtrA2 for promoting cell death.

We have isolated a novel interferon (IFN)-retinoid regulated cell death regulatory protein genes associated with retinoid-IFN-induced mortality (GRIM)-19 earlier. To understand its mechanism of action, we have employed a yeast-two-hybrid screen and identified serine protease HtrA2 as its binding partner. GRIM-19 physically interacts with HtrA2 and augments cell death in an IFN/all-trans retinoic acid (RA)-dependent manner. In the presence of GRIM-19, the HtrA2-driven destruction of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP) is augmented. These interactions were disrupted by an human herpes virus-8 (HHV-8)-coded oncoprotein, vIRF1, and conferred resistance to IFN/RA-induced cell death. These data show a critical role of HtrA2 in a cytokine-induced cell death response for the first time and its inhibition by a viral protein.

A proteomic analysis reveals the loss of expression of the cell death regulatory gene GRIM-19 in human renal cell carcinomas.

Gene associated with retinoid interferon-induced mortality (GRIM)-19, an inhibitor of transcription factor STAT3, was originally identified as a critical regulatory protein in a genetic screen that was designed to identify the gene products necessary for Interferon (IFN)-beta- and retinoic acid-induced cell death. Over expression of GRIM-19 activates cell death. Conversely, inactivation of its expression promotes cell growth. STAT3 is a transcription factor that regulates gene expression in response to multiple extra cellular growth factors. In contrast to its normal feedback inhibition, a constitutive activation of STAT3 has been documented in several tumors. Although many STAT3-inhibitors are described, their relevance to human cancer is unclear. In an attempt to define the molecular alterations associated with human renal cell carcinoma (RCC) using mass spectrometry, we have discovered that expression of GRIM-19 is lost or severely depressed in a number of primary RCC and in some urinogenital tumors. Using an RCC cell line, we show that down regulation of GRIM-19 promotes tumor growth via an augmentation of STAT3-dependent gene expression. These studies for the first time show a tumor-suppressor like activity of GRIM-19.

IFN-gamma inhibits lipopolysaccharide-induced interleukin-1 beta in primary murine macrophages via a Stat1-dependent pathway.

Interleukin-1 (IL-1) plays an important role in host defenses against microbial pathogens. Excessive production of this cytokine, however, may be responsible in part for the lethality observed during sepsis. Our studies show that interferon-gamma (IFN-gamma) downregulates lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1beta) transcription in primary macrophages. This phenomenon does not occur in splenocytes or bone marrow-derived macrophages from signal transducer and activator of transcription (Stat1)-deficient mice, suggesting that Stat1, a transcription factor involved in IFN signaling, plays a critical role in this process. Moreover, nitric oxide (NO) was also involved in the downregulation of LPS-induced IL-1 by IFN, as addition of the inducible nitric oxide synthase (iNOS) inhibitor L-N(6)-(1-iminoethyl)lysine (NIL) negated the effect. Kinetic analysis of IL-1 and IFN levels in LPS-treated mice in vivo suggests that IFN-mediated inhibition of IL-1 might be an important negative feedback mechanism for limiting IL-1 generation in vivo.

Thioredoxin participates in a cell death pathway induced by interferon and retinoid combination.

Interferons (IFNs) and retinoids are potent tumor growth suppressors. We have shown earlier that the IFN-beta and all-trans retinoic acid combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic approach we have recently identified several Genes associated with Retinoid-IFN induced Mortality (GRIM) that mediate the cell death effect of IFN/RA combination. One of the GRIMs, GRIM-12, was identical to human thioredoxin reductase (TR), an enzyme that controls intracellular redox state. To define the participants of TR mediated death pathway we have examined the role of thioredoxin (Trx), its downstream substrate, and its influence on IFN/RA-induced death regulation. Inhibition of the thioredoxin expression by antisense RNA suppressed cell death. Similarly, a mutant Trx1 lacking the critical cysteine residues blocked cell death. In contrast, overexpression of wildtype thioredoxin augmented cell death. This effect of Trx1 was in part due to its ability to augment cell death via caspase-8. The redox inactive Trx1 mutant inhibits the cell death induced by caspase-8 but not caspase-3. These studies identify a novel mechanism of cell death regulation by IFN/RA combination involving redox enzymes.

Modulation of p53 dependent gene expression and cell death through thioredoxin-thioredoxin reductase by the Interferon-Retinoid combination.

We have shown earlier that the IFN-beta and all-trans retinoic acid (RA) combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic technique we have identified several Genes associated with Retinoid-IFN induced Mortality (GRIM). One of the GRIMs was human thioredoxin reductase (TR), a redox enzyme. Since the overexpressed TR augments IFN/RA stimulated cell death, we explored the mechanisms of TR-mediated death. Here we show that TR augments cell death by upregulating the transcriptional activity of p53 tumor suppressor. This process does not involve a physical increase in levels of p53. Using redox inactive mutants of TR and its substrate, thioredoxin (Trx), we demonstrate that IFN/RA-induced regulation of p53 dependent gene expression requires TR and Trx. In contrast-over-expression of wildtype TR or Trx augment the p53 dependent gene expression in response to IFN/RA treatment. Consistent with these results an increased DNA binding activity of p53 was noted in the presence of TR. These studies identify a novel mechanism of p53 mediated cell death regulation involving redox enzymes.

Inositol hexakisphosphate kinase 2 mediates growth suppressive and apoptotic effects of interferon-beta in ovarian carcinoma cells.

Interferons (IFNs) regulate the expression of genes that mediate their antiviral, antitumor, and immunomodulatory actions. We have previously shown that IFN-beta suppresses growth of human ovarian carcinoma xenografts in vivo and induces apoptosis of ovarian carcinoma cells in vitro. To investigate mechanisms of IFN-beta-induced apoptosis we employed an antisense technical knockout approach to identify gene products that mediate cell death and have isolated several regulators of interferon-induced death (RIDs). In this investigation, we have characterized one of the RIDs, RID-2. Sequence analysis revealed that RID-2 was identical to human inositol hexakisphosphate kinase 2 (IP6K2). IP6K2 is post-transcriptionally induced by IFN-beta in ovarian carcinoma cells. A mutant IP6K2 with substitutions in the putative inositol phosphate binding domain abrogates IFN-beta-induced apoptosis. These studies identify a novel function for IP6K2 in cell growth regulation and apoptosis.

Regulation of interferon and retinoic acid-induced cell death activation through thioredoxin reductase.

Interferons (IFNs) and retinoids are potent biological response modifiers. The IFN-beta and all-trans-retinoic acid combination, but not these single agents individually, induces death in several tumor cell lines. To elucidate the molecular basis for these actions, we have employed an antisense knockout approach to identify the gene products that mediate cell death and isolated several genes associated with retinoid-IFN-induced mortality (GRIMs). One of the GRIM cDNAs, GRIM-12, was identical to human thioredoxin reductase (TR). To define the functional relevance of TR to cell death and to define its mechanism of death-modulating functions, we generated mutants of TR and studied their influence on the IFN/RA-induced death regulatory functions of caspases. Wild-type TR activates cell death that was inhibited in the presence of caspase inhibitors or catalytically inactive caspases. A mutant TR, lacking the active site cysteines, inhibits the cell death induced by caspase 8. IFN/all-trans-retinoic acid-induced cytochrome c release from the mitochondrion was promoted in the presence of wild type and was inhibited in the presence of mutant TR. We find that TR modulates the activity of caspase 8 to promote death. This effect is in part caused by the stimulation of death receptor gene expression. These studies identify a new mechanism of cell death regulation by the IFN/all-trans-retinoic acid combination involving redox enzymes.

Mechanism of repression of squamous differentiation marker, SPRR1B, in malignant bronchial epithelial cells: role of critical TRE-sites and its transacting factors.

The overexpression of SPRR1B in bronchial epithelium is a marker for early metaplastic changes and the loss of its expression is associated with an irreversible malignant transformation. In the present study, we have used a model system consisting of normal and malignant bronchial epithelial (BE) cells to elucidate the differential transcriptional control of SPRR1B. SPRR1B expression is either detectable or PMA (phorbol 13-myristate 12-acetate) -inducible in several malignant BE cells including squamous, adeno, small and large cell carcinomas. Loss of SPRR1B expression is correlated well with the lack of strong in vivo protein-DNA interactions at the -152 bp promoter, which contains two functional TRE sites. Even though the basal level AP-1 protein DNA binding pattern is different between normal and malignant cells, PMA significantly enhances Jun and Fos binding to the consensus TRE site in both cell types. Intriguingly, the composition of AP-1 protein binding to the -152 to -86 bp SPRR1B promoter is quite different. In untreated cells, SPRR1B promoter is predominantly occupied by JunD and Fra2. PMA significantly induced binding of JunB and Fra1 in normal cells, while JunB and Fra2 bound to TREs in the malignant cells. Overexpression of fra1 in malignant cells significantly enhanced SPRR1B promoter activity. In contrast, overexpression of fra2, but not fra1, strongly reduced both basal and PMA-inducible promoter activities in normal cells. Together, these results indicate that either temporal expression and/or differential activation of AP-1 proteins, especially Fra1 and Fra2, might contribute to the dysregulation of terminal differentiation marker, SPRR1B, expression in various BE cells.

ERK1 and ERK2 activate CCAAAT/enhancer-binding protein-beta-dependent gene transcription in response to interferon-gamma.

Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-beta (C/EBP-beta) induces gene transcription through a novel IFN response element called the gamma-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626-12632. Here, we describe a new IFN-gamma-stimulated pathway that operates C/EBP-beta-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-gamma to stimulate C/EBP-beta-dependent expression. Sustained ERK activation directly correlated with C/EBP-beta-dependent gene expression in response to IFN-gamma. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-gamma-stimulated gene induction through the gamma-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-beta-induced gene expression required STAT1, but not JAK1. A C/EBP-beta mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-beta to IFN-gamma signaling through ERKs.

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to all-trans-retinoic acid.

Several signaling pathways are activated by all-trans-retinoic acid (RA) to mediate induction of differentiation and apoptosis of malignant cells. In the present study we provide evidence that the p38 MAP kinase pathway is activated in a RA-dependent manner in the NB-4, acute pro-myelocytic leukemia, and the MCF-7, breast carcinoma, cell lines. RA treatment of cells induces a time- and dose-dependent phosphorylation of p38, and such phosphorylation results in activation of its catalytic domain. p38 activation is not inducible by RA in a variant NB-4 cell line, NB-4.007/6, which is resistant to the effects of RA, suggesting a role for this pathway in the induction of RA responses. Our data also demonstrate that the small G-protein Rac1 is activated by RA and functions as an upstream regulator of p38 activation, whereas the MAPKAPK-2 serine kinase is a downstream effector for the RA-activated p38. To obtain information on the functional role of the Rac1/p38/MAPKAPK-2 pathway in RA signaling, the effects of pharmacological inhibition of p38 on RA-induced gene transcription and cell differentiation were determined. Our results indicate that treatment of cells with the SB203580 inhibitor does not inhibit RA-dependent gene transcription via retinoic acid response elements or induction of Stat1 protein expression. However, treatment with SB203580 or SB202190 strongly enhances RA-dependent induction of cell differentiation and RA-regulated growth inhibitory responses. Altogether, our findings demonstrate that the Rac1/p38 MAP kinase pathway is activated in a RA-dependent manner and exhibits negative regulatory effects on the induction of differentiation.

Interleukin-6 modulates interferon-regulated gene expression by inducing the ISGF3 gamma gene using CCAAT/enhancer binding protein-beta(C/EBP-beta).

Although interleukin-6 (IL-6) alone does not induce the expression of IFN stimulated genes (ISG), a low dose priming of cells with IL-6 strongly enhances the cellular responses to interferon-alpha (IFN-alpha). This effect of IL-6 is not due to superstimulation of the JAK-STAT pathway. Rather, IL-6 induces expression of ISGF3 gamma (p48), a subunit of the multimeric transcription factor ISGF3. As a result IFN-alpha robustly activates gene transcription in IL-6 primed cells. We have shown earlier that the transcription of ISGF3 gamma gene is regulated through a novel element GATE (gamma-IFN activated transcriptional element). We show here IL-6 induces the ISGF3 gamma gene through GATE. Transcription factor C/EBP-beta is required for inducing ISGF3 gamma gene expression through GATE. A mutant C/EBP-beta inhibits the IL-6 inducible ISGF3 gamma gene expression through GATE. Together, these results establish a molecular basis for the synergy between IFNs and IL-6.

Identification of GRIM-19, a novel cell death-regulatory gene induced by the interferon-beta and retinoic acid combination, using a genetic approach.

We show here that the combination of interferon-beta (IFN-beta) and all-trans-retinoic acid (RA) induces the death of tumor cells. To understand the molecular basis for synergistic growth-suppressive action and to identify the gene products that participate in this process, we have employed an antisense knock-out technique. This approach permits the isolation of cell death-associated genes based on their selective inactivation by overexpression of antisense cDNAs. Because the antisense mRNA inactivates gene expression of death-specific genes, transfected cells survive in the presence death inducers. Several Genes associated with Retinoid-IFN-induced Mortality (GRIM) were identified using this approach. Here we report the isolation of a novel GRIM gene, GRIM-19. This 552-base pair cDNA encodes a 16-kDa protein. Antisense expression of GRIM-19 confers a strong resistance against IFN/RA-induced death by reducing the intracellular levels of GRIM-19 protein. Overexpression of GRIM-19 enhances cell death in response to IFN/RA. GRIM-19 is primarily a nuclear protein whose expression is induced by the IFN/RA combination. Together, our studies identify a novel cell death-regulatory molecule.

Phorbol ester-induced expression of airway squamous cell differentiation marker, SPRR1B, is regulated by protein kinase Cdelta /Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway.

The transcriptional induction of SPRR1B by phorbol 12-myristate 13-acetate (PMA) is mainly mediated by the first -152-base pair 5'-flanking region containing two functional AP-1 sites. In this study, we have analyzed the signaling pathways that mediate the induction in tracheobronchial epithelial cells. PKC inhibitor ablated PMA-stimulated expression of endogenous SPRR1B and reporter gene expression driven by SPRR1B promoter. PKC activator promoted the transcription. The dominant negative protein kinase Cdelta (dn-PKCdelta) and rottlerin (PKCdelta inhibitor) completely suppressed PMA-stimulated promoter activity. dn-Ras or dn-MEKK1 inhibited PMA-stimulated promoter activity, while their corresponding constitutively active mutants augmented it. dn-c-Raf-1 did not have any effect on reporter gene expression. Since MEKK1 activates multiple parallel pathways, we examined involvement of JNK/SAPK, p38, and MKK1 in promoter regulation. Co-expression of the dominant negative forms of MKK4, MKK7, JNK/SAPK, MKK3, MKK6, or p38alpha did not suppress PMA-stimulated reporter gene expression. However, MKK1 inhibitors UO126 and PD98095 suppressed gene expression. Consistent with this, expression of dn-MKK1 strongly suppressed PMA-stimulated promoter activity, while the constitutively active MKK1 augmented it. However, MKK1-mediated induction of SPRR1B probably does not depend on extracellular signal-regulated kinases 1 and 2, suggesting the requirement of another kinase(s). dn-c-Jun mutants abolished PMA-stimulated expression supporting an important role for AP-1 proteins in SPRR1B expression. Together, these results suggest that a PKCdelta/Ras/MEKK1/MKK1-dependent/AP-1 pathway regulates the PMA-inducible expression of the SPRR1B in tracheobronchial epithelial cells.

Chromosomal localization of human GRIM-19, a novel IFN-beta and retinoic acid-activated regulator of cell death.

Apoptosis is a tightly regulated mechanism that controls the proliferation of cells in metazoans. In mammals, multiple genes are required to regulate cell death. We have employed a gene expression knockout technique to isolate cell death-related genes. One such gene, gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), is essential for tumor cell death induced by interferon-beta (IFN-beta) and retinoic acid (RA). Here, we describe the localization of GRIM-19 to human chromosome 19p13.2. This region is essential for prostate tumor suppression. Together with its death-inductive role in the IFN-retinoid-regulated pathways and the tumor-suppressive function of this locus, the data suggest that GRIM-19 may be a novel tumor suppressor.

Interferons and cell growth control.

Cytokines modulate cell growth, differentiation, and immune defenses in the vertebrates. Interferons (IFNs) are a unique class of cytokines that stimulate antiviral, antitumor and antigen presentation by inducing the expression of several cellular genes. Recent studies have identified a novel gene regulatory pathway activated by IFNs, which serves as a paradigm for most cytokine signal transduction pathways. A number of genes induced by IFNs participate in cell growth regulation and apoptosis. These include novel tumor suppressor genes. Although discovered as IFN-regulated factors, deletions of these genes cause leukemias in experimental models and in human patients. Genetic approaches have identified several novel regulators of apoptosis. Studies on the mechanism of action of these growth regulatory molecules are not only useful in identifying novel targets for the development of therapeutics but also help understand the molecular basis for loss of cell growth control and resistance to IFNs. This review focuses on the functions and roles of IFN regulated factors in cell growth control and mechanisms of disruption of IFN action in cancer cells.

CCAAT/enhancer-binding protein-beta regulates interferon-induced transcription through a novel element.

We have described previously a novel interferon (IFN)-responsive cis-acting enhancer element called gamma-IFN-activated transcriptional element (GATE). GATE is distinct from the known IFN-stimulated elements and binds to novel transacting factors. To identify the gamma-IFN-responsive transacting factors that interact with GATE, we have screened a cDNA expression library derived from IFN-gamma-stimulated murine macrophage cell line and isolated three different cDNAs. Among these is a gene coding for the pleiotropic transcription factor, CCAAT/enhancer-binding protein-beta (C/EBP-beta). We report here that the gene for C/EBP-beta binds to GATE and induces gene expression. A mutant C/EBP-beta interferes with the IFN-gamma-stimulated transcription of the ISGF3gamma (p48) promoter. Other members of the C/EBP family do not cause these effects. Interestingly, the expression of C/EBP-beta, not the other members of its family, is induced by IFN-gamma. These studies thus identify a novel role for C/EBP-beta in the IFN-signaling pathways.