Increased carcinogenic potential of myeloid tumor cells induced by aberrant TGF-beta1-signaling and upregulation of cathepsin B

The TGF-beta signaling pathways are implicated in cancer. Cysteine cathepsins can contribute to the carcinogenic potential of tumor cells. The aim of this study was to investigate the regulation of cysteine cathepsin expression by TGF-beta1 and the functional implications in tumor cells. We found an upregulation of cathepsin B (CathB, 2- to 5-fold) in different myeloid tumor cells (THP-1, MonoMac-1, MonoMac-6) after incubation with TGF-beta1. No upregulation was found in monocytes, and there was suppression of CathB expression in epithelial tumor cells (A549). Increased cathepsin B activity led to enhanced carcinogenic potential, which was reflected by increased migration and invasion of the cells and resistance to inhibitor-induced apoptosis. Analysis of the TGF-beta signaling pathways showed no alterations in TGF-beta/BMP receptor expression or SMAD2/3 phosphorylation, and no influence of MAP kinase pathways. However, a reduction in SMAD1 expression was detected. The lack of BMP action on cysteine cathepsin expression in myeloid tumor cells, but not in epithelial tumor cells, suggests a defect in the Smad1/Smad5 pathway. We located a related TGF-beta1-responsive element within the first intron of the CathB gene. In conclusion, alterations in the TGF-beta1 signaling pathway lead to upregulation of CathB, which contributes to the carcinogenic potential of tumor cells.     

Targeted mutations of transforming growth factor-beta genes reveal important roles in mouse development and adult homeostasis.

Transforming growth factors-beta (TGF-beta) are multifunctional molecules with profound biological effects in many developmental processes including regulation of cell proliferation, differentiation, cell adhesion, skeletal development, haematopoiesis, inflammatory responses, and wound healing. To learn about the role of TGF-beta in vivo, phenotypes of targeted mutations of molecules within the TGF-beta signalling pathway, TGF-beta1, -beta2, -beta3, TGF-beta receptor (TbetaR-II) and the signalling molecules SMAD2, SMAD3 and SMAD4, are discussed in this review. The three individual TGF-beta mutants show distinct and only partially overlapping phenotypes. In mice, targeted disruption of the TGF-beta1 gene results in diffuse and lethal inflammation about 3 weeks after birth, suggesting a prominent role of TGF-beta in the regulation of immune cell proliferation and extravasation into tissues. However, just half of the TGF-beta1 (-/-) conceptuses actually reach partuition due to defective haematopoiesis and endothelial differentiation. Targeted disruption of both TGF-beta2 and TGF-beta3 genes results in perinatal lethality. TGF-beta2 null mice exhibit a broad range of developmental defects, including cardiac, lung, craniofacial, limb, eye, ear and urogenital defects, whereas TGF-beta3 gene ablation results exclusively in defective palatogenesis and delayed pulmonary development. The TbetaR-II null phenotype closely resembles that of TGF-beta1 (-/-) conceptuses, which die in utero by E10.5. Loss of SMAD2 or SMAD4 results in related phenotypes: the mutants fail to form an organized egg cylinder, lack mesoderm required for gastrulation and die prior to E8.5. Together, gene ablation within the TGF-beta signalling pathway supports the notion of a prominent role of TGF-beta during development.     

E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.     

Transforming growth factor-beta stimulates cyclin D1 expression through activation of beta-catenin signaling in chondrocytes

Transforming growth factor-beta (TGF-beta) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-beta rapidly induced beta-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-beta-increased beta-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3(-/-) chondrocytes treated with TGF-beta. SMAD3 inhibited beta-transducin repeat-containing protein-mediated degradation of beta-catenin and immunoprecipitated with beta-catenin following TGF-beta treatment. Both SMAD3 and beta-catenin co-localized to the nucleus after TGF-beta treatment. Although both TGF-beta and beta-catenin stimulated cyclin D(1) expression in chondrocytes, the effect of TGF-beta was inhibited with beta-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-beta stimulates cyclin D(1) expression at least in part through activation of beta-catenin signaling.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Effect of human papillomavirus type 16 oncogenes on MAP kinase activity.

The mitogen-activated protein (MAP) kinase signal transduction pathway is an intracellular signaling cascade which mediates cellular responses to growth and differentiation factors. The MAP kinase pathway can be activated by a wide range of stimuli dependent on the cell types, and this is normally a transient response. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells in part by prolonging the activated stage of components within this signaling pathway. The human papillomavirus (HPV) oncogenes E6 and E7 play an essential role in the in vitro transformation of primary human keratinocytes and rodent cells. The HPV type 16 E5 gene has also been shown to have weak transforming activity and may enhance the epidermal growth factor (EGF)-mediated signal transduction to the nucleus. In the present study, we have investigated the effects of the oncogenic HPV type 16 E5, E6, and E7 genes on the induction of the MAP kinase signaling pathway. The E5 gene induced an increase in the MAP kinase activity both in the absence and in the presence of EGF. In comparison, the E6 and E7 oncoproteins do not alter the MAP kinase activity or prolong the MAP kinase activity induced with EGF. These findings suggest that E5 may function, at least in part, to enhance the cell response through the MAP kinase pathway. However, the transforming activity of E6 and E7 is not associated with alterations in the MAP kinase pathway. These findings are consistent with E5 enhancing the response to growth factor stimulation.     

The E6 oncoprotein from HPV16 enhances the canonical Wnt/β-catenin pathway in skin epidermis in vivo

The contribution of the Wnt signaling pathway to human papilloma virus (HPV)-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk HPVs (HR-HPV), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the PDZ-binding domain of E6 protein. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2(+/LacZ) mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellular β-catenin-responsive genes, and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2(+/LacZ). Conversely, cotransfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that used a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only.     

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

NF-kappaB protects human papillomavirus type 38 E6/E7-immortalized human keratinocytes against tumor necrosis factor alpha and UV-mediated apoptosis

Constitutive activation of NF-κB signaling is a key event in virus- and non-virus-induced carcinogenesis. We have previously reported that cutaneous human papillomavirus type 38 (HPV38) displays transforming properties in in vitro and in vivo experimental models. However, the involvement of NF-κB signaling in HPV38-induced cell growth transformation remains to be determined. In this study, we showed that HPV38 E6 and E7 activate NF-κB and that inhibition of the pathway with the IκBα superrepressor sensitizes HPV38E6E7-immortalized human keratinocytes to tumor necrosis factor alpha (TNF-α)- and UVB radiation-mediated apoptosis. Accordingly, inhibition of NF-κB signaling resulted in the downregulation of NF-κB-regulated antiapoptotic genes, including cIAP1, cIAP2, and xIAP genes. These findings demonstrate a critical role of NF-κB activity in the survival of HPV38E6E7-immortalized human keratinocytes exposed to cytokine or UV radiation. Our data provide additional evidence for cooperation between beta HPV infection and UV irradiation in skin carcinogenesis.     

The HPV16 E6 Oncoprotein Causes Prolonged Receptor Protein Tyrosine Kinase Signaling and Enhances Internalization of Phosphorylated Receptor Species

The high-risk human papillomavirus (HPV) E6 proteins are consistently expressed in HPV-associated lesions and cancers. HPV16 E6 sustains the activity of the mTORC1 and mTORC2 signaling cascades under conditions of growth factor deprivation. Here we report that HPV16 E6 activated mTORC1 by enhanced signaling through receptor protein tyrosine kinases, including epidermal growth factor receptor and insulin receptor and insulin-like growth factor receptors. This is evidenced by sustained signaling through these receptors for several hours after growth factor withdrawal. HPV16 E6 increased the internalization of activated receptor species, and the signaling adaptor protein GRB2 was shown to be critical for HPV16 E6 mediated enhanced EGFR internalization and mTORC1 activation. As a consequence of receptor protein kinase mediated mTORC1 activation, HPV16 E6 expression increased cellular migration of primary human epithelial cells. This study identifies a previously unappreciated mechanism by which HPV E6 proteins perturb host-signaling pathways presumably to sustain protein synthesis during the viral life cycle that may also contribute to cellular transforming activities of high-risk HPV E6 proteins.     

Differential regulation of cutaneous oncoprotein HPVE6 by wtp53, mutant p53R248W and ΔNp63α is HPV type dependent

UV exposure and p53 mutations are major factors in non-melanoma skin cancer, whereas a role for HPV infections has not been defined. Previous data demonstrated the wtp53-mediated degradation of cutaneous HPV20E6 by caspase-3. ΔNp63α and hot-spot mutant p53R248W conveyed a protective effect on HPV20E6 under these conditions. We demonstrate a differential regulation by wtp53 of the E6 genes of cutaneous types HPV4, HPV5, HPV7, HPV27, HPV38, HPV48, HPV60 and HPV77. Caspase- or proteasome-mediated down-regulation was HPV type dependent. Mutant p53R248W up-regulated expression of all these E6 proteins as did ΔNp63α except for HPV38E6 which was down-regulated by the latter. None of these cellular proteins affected HPV41E6 expression. Ectopic expression of both mutp53R248W and ΔNp63α in the normal NIKS keratinocyte cell line harbouring endogenous p53 and p63however led to a down-regulation of HPV20E6. We demonstrate that HPV20E6 expression in these cells is modulated by additional, yet unidentified, cellular protein(s), which are not necessarily involved in apoptosis or autophagy. We further demonstrate proliferation of HPV20E6-expressing keratinocytes. Levels of proteins involved in cell cycle control, cyclin-D1, cdk6 and p16(INK4a), phosphorylated pRB, as well as c-Jun and p-c-Jun, were all increased in these cells. HPV20E6 did not compete for the interaction between p16(INK4a) with cyclin-D1 or cdk6. Phosphorylation of pRB in the HPV20E6 expressing cells seems to be sufficient to override the cytokenetic block induced by the p16(INK4a)/pRB pathway. The present study demonstrates the diverse influence of p53 family members on individual cutaneous HPVE6 proteins. HPV20E6 expression also resulted in varying protein levels of factors involved in proliferation and differentiation.     

Influence of Human Papillomavirus E7 Oncoprotein on Maturation and Function of Plasmacytoid Dendritic Cells In Vitro

The major difficulties of human papillomavirus(HPV) treatment are its persistence and recurrence. The HPV E7 oncoprotein-loaded dendritic cells have been evaluated as cellular vaccine in previous reports. Plasmacytoid dendritic cells(pDCs) play an essential role of connecting the innate immune response and adaptive immune response in the immune system. But they function in HPV E7 loading is unclear. To investigate whether loading of the HPV type 6b, 11, and 16 E7 proteins affects the activity of pDCs, human peripheral blood-separated pDCs and mouse bone marrow-derived pDCs were pulsed with the HPV E7 proteins. The expression levels of CD40, CD80, CD86, and MHC II were significantly upregulated in pDCs upon HPV 6b/11 E7 protein pulse. The secretion and gene expression of type I IFN and IL-6 were both upregulated by HPV 6b/11 E7 proteins, more significant than HPV 16 E7 protein. The expression of essential factors of TLR signaling pathway and JNK/p38 MAP kinase signaling pathway were all increased in HPV 6b/11 E7 proteins pulsed pDCs. Our results suggest that HPV E7 proteins could promote the differentiation and maturation of pDCs and activate the TLR and MAPK pathway to induce host innate immune response. It might be conducive to explore novel immunotherapy targeting HPV infection with HPV E7 loaded pDC.