Cytokine-based transformation of immune surveillance into tumor-promoting inflammation.

During the last decade, it has become clear that the mammalian immune system is able to recognize and partially suppress nascent tumors. Human T cells specific to oncogenes and onco-fetal antigens are present in human cancer patients and their tumors. At the same time, molecular links between tumor-associated inflammation and tumor progression have been uncovered, providing an explanation for the long recognized epidemiological link between inflammation and cancer. The synopsis of these findings suggests a new interpretation of tumor immunity. It appears that antigen recognition or antigen-specific T-cell expansion at large is not as profoundly impaired in tumor patients as the correct polarization, the survival and the effector function of tumor-infiltrating T cells. This review will focus on pro-inflammatory cytokines likely to contribute to the deregulation of tumor-specific immunity and its consequences.

TGF-beta inhibits p70 S6 kinase via protein phosphatase 2A to induce G(1) arrest.

On TGF-beta binding, the TGF-beta receptor directly phosphorylates and activates the transcription factors Smad2/3, leading to G(1) arrest. Here, we present evidence for a second, parallel, TGF-beta-dependent pathway for cell cycle arrest, achieved via inhibition of p70(s6k). TGF-beta induces association of its receptor with protein phosphatase-2A (PP2A)-Balpha. Concomitantly, three PP2A-subunits, Balpha, Abeta, and Calpha, associate with p70(s6k), leading to its dephosphorylation and inactivation. Although either pathway is sufficient to induce G(1) arrest, abrogation of both, the inhibition of p70(s6k), and transcription through Smad proteins is required for release of epithelial cells from TGF-beta-induced G(1) arrest. TGF-beta thereby modulates the translational and posttranscriptional control of cell cycle progression.

TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis.

BACKGROUND: Invasive growth of epithelial tumor cells, a major cause of death from cancer in humans, involves loss of epithelial polarity and dedifferentiation. Transforming growth factor beta (TGFbeta) is regarded as a major tumor suppressor during early tumor development because it inhibits cell-cycle progression and tumor growth. Many dedifferentiated, late-stage tumors are resistant to growth inhibition by TGFbeta, however, and even secrete TGFbeta. In line with this, TGFbeta is involved in angiogenesis, wound healing and epithelial-mesenchymal transition (EMT) during development. Ha-Ras-transformed mammary epithelial cells (EpRas) undergo TGFbeta-induced EMT maintained via a TGFbeta autocrine loop. Thus, we have analyzed whether signal transduction by the TGFbeta receptor (TGFbetaR) is required for local tumor cell invasion and metastasis. RESULTS: A dominant-negative type II TGFbetaR (TGFbetaRII-dn) was expressed using retroviral vectors in EpRas cells and highly metastatic mesenchymal mouse colon carcinoma cells (CT26). In both cell types, TGFbetaRII-dn blocked TGFbetaR signaling and heavily delayed tumor formation. In EpRas cells, TGFbetaRII-dn prevented EMT. In the dedifferentiated mesenchymal CT26 cells, TGFbetaRII-dn caused mesenchymal-to-epithelial transition and inhibited their in vitro invasiveness in several assays. In addition, TGFbetaRII-dn completely abolished metastasis formation by CT26 cells. Furthermore, several human carcinoma lines lost in vitro invasiveness when treated with neutralizing TGFbeta antibodies or soluble receptor variants. Finally, human colon carcinoma cells (hnPCC) expressing a mutated, non-functional TGFbetaRII were non-invasive in vitro, a defect restored by re-expressing wild-type TGFbetaRII. CONCLUSIONS: Cell-autonomous TGFbeta signaling is required for both induction and maintenance of in vitro invasiveness and metastasis during late-stage tumorigenesis. TGFbetaRII therefore represents a potential target for therapeutical intervention in human tumorigenesis.

Functional analysis of the human immunodeficiency virus type 1 Rev protein oligomerization interface.

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.

Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5.

It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.

TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells.

Metastasis of epithelial tumor cells can be associated with the acquisition of fibroblastoid features and the ability to invade stroma and blood vessels. Using matched in vivo and in vitro culture systems employing fully polarized, mammary epithelial cells, we report here that TGF-beta1 brings about these changes in Ras-transformed cells but not in normal cells. When grown in collagen gels in the absence of TGF-beta, both normal and Ras-transformed mammary epithelial cells form organ-like structures in which the cells maintain their epithelial characteristics. Under these conditions, treatment of normal cells with TGF-beta results in growth arrest. The same treatment renders Ras-transformed epithelial cells fibroblastoid, invasive, and resistant to growth inhibition by TGF-beta. After this epithelial-fibroblastoid conversion, the Ras-transformed cells start to secrete TGF-beta themselves, leading to autocrine maintenance of the invasive phenotype and recruitment of additional cells to become fibroblastoid and invasive. More important, this cooperation of activated Ha-Ras with TGF-beta1 is operative during in vivo tumorigenesis and, as in wound healing processes, is dependent on epithelial-stromal interactions.

The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions.

Members of the epidermal growth factor (EGF) receptor family are known to be specifically involved in mammary carcinogenesis. As a nuclear target of activated receptors, we examined c-Jun in mammary epithelial cells. For this, we used a c-JunER fusion protein which was tightly controlled by estrogen. Activation of the JunER by hormone resulted in the transcriptional regulation of a variety of AP-1 target genes. Hormone-activated JunER induced the loss of epithelial polarity, a disruption of intercellular junctions and normal barrier function and the formation of irregular multilayers. These changes were completely reversible upon hormone withdrawal. Loss of epithelial polarity involved redistribution of both apical and basolateral proteins to the entire plasma membrane. The redistribution of E-cadherin and beta-catenin was accompanied by a destabilization of complexes formed between these two proteins, leading to an enrichment of beta-catenin in the detergent-soluble fraction. Uninduced cells were able to form three-dimensional tubular structures in collagen I gels which were disrupted upon JunER activation, leading to irregular cell aggregates. The JunER-induced disruption of tubular structures was dependent on active signaling by growth factors. Moreover, the effects of JunER could be mimicked in normal cells by the addition of acidic fibroblast growth factor (aFGF). These data suggest that a possible function of c-Jun in epithelial cells is to modulate epithelial polarity and regulate tissue organization, processes which may be equally important for both normal breast development and as initiating steps in carcinogenesis.

Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

Expression of the different viral mRNAs of human papilloma virus 6 in a squamous-cell carcinoma of the bladder and the cervix.

The prevalence of HPV6 and HPV11 in benign condylomata or mild dysplasias has led to the view of HPV6/11 as rather harmless viruses in relation to carcinogenesis. However, the detection of HPV6/11 DNA in a number of individual cases of squamous-cell carcinomas of the anogenital/urinary tract could also point to a possible contribution of these viruses in the development of certain malignancies. Recently we have shown that the transcription of the E6 and E7 genes of HPV6 in benign anogenital condylomata is strictly confined to the basal cell layers of the epithelium, which express c-fos mRNA. This report describes the in situ hybridization analysis of individual mRNA species of HPV6 in 2 malignant tumours. A consistent feature of both carcinomas was the lack of detectable amounts of E6 mRNA, while the E7 mRNA was the major transcript observed. In situ hybridization with a riboprobe for c-fos revealed an expression pattern similar to that detected with the E7 probe. Hybridization with a probe specific for mRNA with a coding potential for a full-length E2 protein yielded weak signals in both carcinomas. Using restriction-enzyme analysis, we compared the long control region of HPV6 amplified by polymerase chain reaction from both tumours with already known HPV6 subtypes. In contrast to previous reports suggesting a correlation between genetic alterations in the long control region of HPV6 and increased malignant behaviour, our data do not support this hypothesis.

Human papillomavirus type 6 in squamous cell carcinoma of the bladder and cervix.

Human papillomavirus (HPV) types 16 and 18 are the most frequent genotypes identified in genital malignancies, while HPV types 6 and 11 are found predominantly in condylomas and low-grade dysplasias. It is thought that HPV types 16 and 18 represent high-risk genotypes, while HPV types 6 and 11 rarely, if ever, participate in the development of malignant tumors. In a series of over 300 invasive tumors of the lower genital tract analyzed for the presence of HPV three have been found to contain HPV type 6 DNA: two invasive squamous cell carcinomas of the cervix and one squamous cell carcinoma of the bladder. Human papillomavirus type 6 was the only HPV type detected in these tumor DNAs by Southern blot hybridization and by the polymerase chain reaction using both consensus and type-specific primers. In situ hybridization using whole genomic RNA probes localized viral DNA to tumor cells. Although extensive virologic and epidemiologic studies conducted in the last decade indicate that HPV types 16 and 18 are more likely to be associated with high-grade dysplasias and invasive cancer, HPV type 6 may not be as innocuous as previously supposed.

Transcription of the E6 and E7 genes of human papillomavirus type 6 in anogenital condylomata is restricted to undifferentiated cell layers of the epithelium.

The E6 and E7 genes of human genital papillomaviruses (HPVs) appear to transform cells by different mechanisms. They seem to act synergistically but are not equally important when tested under diverse experimental conditions. We were therefore tempted to investigate the E6- and E7-specific transcription pattern in HPV6-infected condylomas separately, by in situ hybridization. Recent studies have identified three promoters within the E6-E7 region of HPV6 and HPV11 by applying S1, exonuclease VII, and cDNA analyses. On the basis of these data, we cloned subgenomic fragments of HPV6 into plasmid pBS to obtain riboprobes that differentiated between transcripts starting upstream of the E6 and E7 open reading frames, respectively. These different species of mRNAs were analyzed in serial thin sections of eight HPV6-positive anogenital condylomas. The E6 probe (nucleotides 7862 to 241) led to weak signals within the basal layer. In three cases, rather strong signals were confined to a few basal cells. The E7 probe (nucleotides 242 to 534) gave rise to a more pronounced labeling of all cells within the two to three lowest epidermal layers. In situ hybridization with a riboprobe for human c-fos revealed an expression pattern similar to that observed with the E7 probe. In contrast to the preferential expression of the transforming E6 and E7 genes in the lower epithelium, the major transcriptional activity of the virus was detected in the middle and upper third by probes colinear with the 3' moiety of the early region.