Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.

The EGF‐receptor (EGF‐R) is a transmembrane glycoprotein with intrinsic protein tyrosine kinase (TK) activity. To explore the importance of the receptor TK in the action of EGF, we have used transfected NIH‐3T3 cells expressing either the normal human EGF‐R or a receptor mutated at Lys721, a key residue in the presumed ATP‐binding region. The wild‐type receptor responds to EGF by causing inositol phosphate formation, Ca2+ influx, activation of Na+/H+ exchange and DNA synthesis. In contrast, the TK‐deficient mutant receptor fails to evoke any of these responses. It is concluded that activation of the receptor TK is a crucial signal that initiates the multiple post‐receptor effects of EGF leading to DNA synthesis. Furthermore, the results suggest that tyrosine phosphorylation plays a role in the activation of the phosphoinositide signalling system.

The human papillomavirus (HPV) E6/E7 oncogenes play a crucial role in the HPV-induced carcinogenesis. In this study, the authors investigated whether silencing of endogenous HPV E6/E7 expression may influence the contents or amounts of extracellular microvesicles (eMVs) released from HPV-positive cancer cells. It was found that eMVs secreted from HeLa cells are enriched for Survivin protein. RNA interference studies revealed that maintenance of both intracellular and microvesicular Survivin amounts was strongly dependent on continuous E6/E7 expression. This indicates that intracellular HPV activities are translated into visible alterations of protein contents in eMVs. Besides Survivin, eMVs from HeLa cells contain additional members of the inhibitor of apoptosis protein (IAP) family (XIAP, c-IAP1 and Livin). In contrast, no evidence for the presence of the HPV E6 and E7 oncoproteins in eMVs was obtained. Moreover, it was found that silencing of HPV E6/E7 expression led to a significant increase of exosomes-representing eMVs of endocytic origin-released from HeLa cells. This effect was associated with the reinduction of p53, stimulation of the p53 target genes TSAP6 and CHMP4C that can enhance exosome production and induction of senescence. Taken together, these results show that silencing of HPV E6/E7 oncogene expression profoundly affects both the composition and amounts of eMVs secreted by HPV-positive cancer cells. This indicates that HPVs can induce molecular signatures in eMVs that may affect intercellular communication and could be explored for diagnostic purposes.Specific types of human papillomaviruses (HPVs), such as HPV16 and HPV18, cause cervical cancer and are closely linked to the development of additional human malignancies in the oropharyngeal and anogenital regions. 1 The viral E6 and E7 oncoproteins are crucial both for the HPV-associated induction of transformation and for the maintenance of the tumorigenic phenotype of HPV-positive cervical cancer cells. 2,3 E6 and E7 dysregulate intracellular pathways involved in the control of cellular proliferation, apoptosis and genetic stability. For example, E6 induces the proteolytic degradation of the p53 tumor suppressor protein 4 and stimulates telomerase activity, 5 whereas E7 interferes with the activity of the retinoblastoma tumor suppressor protein, pRb, and other pocket proteins. 6 In contrast to the increasing understanding of the intracellular activities of the viral E6/E7 oncogenes, surprisingly little is known about possible effects on the intercellular communication of HPV-positive cancer cells.Extracellular microvesicles (eMVs) include exosomes that are small vesicles (50-100 nm in diameter) of endosomal origin secreted by a variety of cells, including tumor cells. 7 Exosomes have recently gained much interest in oncology, particularly due to three properties: (i) exosomes secreted from tumor cells can suppress the immune response toward the tumor, 8,9 (ii) tumor cell-derived exosomes can accelerate tumor growth and invasiveness by horizon...

In vitro site-directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF) -

Epidermal growth factor (EGF)‐receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH‐3T3 cells lacking endogenous EGF‐receptors. Kinetic parameters of the kinase of wild‐type and mutant receptors were compared. Both wild‐type and mutant EGF‐receptors had a Km(ATP) 1‐3 microM for the autophosphorylation reaction, and a Km(ATP) of 3‐7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF‐receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF‐receptor (KGSTAENAEYLRV) was phosphorylated by wild‐type receptor with a Km of 110‐130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF‐receptors phosphorylated the peptide substrate with a Km of 70‐100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c‐src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild‐type receptor to 890 microM for mutant receptor and for c‐src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650‐750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGFinsulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that defime kinase specificity, mitogenic and transforming potential, and receptor routing.

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The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.