Identification of E6/E7-Dependent MicroRNAs in HPV-Positive Cancer Cells.

Oncogenic types of human papillomaviruses (HPVs) are closely linked to the development of anogenital and head and neck cancers . The expression of the viral E6 and E7 genes is crucial for the transforming activities of HPVs. There is accumulating evidence that the HPV E6/E7 oncogenes can profoundly affect the cellular microRNA (miRNA) composition. Since alterations of miRNA expression levels can contribute to cancer development and maintenance, it will be important to understand in depth the crosstalk between the HPV oncogenes and the cellular miRNA network . Here, we describe a method to identify E6/E7-dependent intracellular miRNAs by small RNA deep sequencing , upon silencing of endogenous E6/E7 expression in HPV-positive cancer cells in vitro. In addition, we provide a protocol to identify E6/E7-dependent miRNA alterations in exosomes that are secreted by HPV-positive cancer cells in vitro.

Viral E6/E7 oncogene and cellular hexokinase 2 expression in HPV-positive cancer cell lines.

Oncogenic types of human papillomaviruses (HPVs) are major human carcinogens. Cancer cells typically exhibit metabolic alterations which support their malignant growth. These include an enhanced rate of aerobic glycolysis ('Warburg effect') which in cancer cells is often linked to an increased expression of the rate-limiting glycolytic enzyme Hexokinase 2 (HK2). Intriguingly, recent studies indicate that the HPV E6/E7 oncogenes cause the metabolic reprogramming in HPV-positive cancer cells by directly upregulating HK2 expression. Notably, however, these results were obtained upon ectopic overexpression of E6/E7. Here, we investigated whether HK2 levels are affected by the endogenous E6/E7 amounts present in HPV-positive cancer cell lines. RNA interference analyses reveal that the sustained E6/E7 expression is critical to maintain HK2 expression levels in HeLa cells. Mechanistically, this effect is linked to the E6/E7-dependent upregulation of HK2-stimulatory MYC expression and the E6/E7-induced downregulation of the HK2-inhibitory micro(mi)RNA miR-143-3p. Importantly, however, a stimulatory effect of E6/E7 on HK2 expression was observed only in HeLa among a panel of 8 different HPV-positive cervical and head and neck cancer cell lines. Thus, whereas these results support the notion that E6/E7 can increase HK2 expression, they argue against the concept that the viral oncogenes, at endogenous expression levels, commonly induce the metabolic switch of HPV-positive cancer cells towards aerobic glycolysis by directly or indirectly stimulating HK2 expression.

Intracellular Analysis of the Interaction between the Human Papillomavirus Type 16 E6 Oncoprotein and Inhibitory Peptides.

Oncogenic types of human papillomaviruses (HPVs) cause cervical cancer and other malignancies in humans. The HPV E6 oncoprotein is considered to be an attractive therapeutic target since its inhibition can lead to the apoptotic cell death of HPV-positive cancer cells. The HPV type 16 (HPV16) E6-binding peptide pep11, and variants thereof, induce cell death specifically in HPV16-positive cancer cells. Although they do not encompass the LxxLL binding motif found in cellular HPV16 E6 interaction partners, such as E6AP, the pep11 variants strongly bind to HPV16 E6 by contacting the recently identified E6AP binding pocket. Thus, these peptides can serve as prototype E6-inhibitory molecules which target the E6AP pocket. We here analyzed their intracellular interaction with HPV16 E6. By comprehensive intracellular binding studies and GST pull-down assays, we show that E6-binding competent pep11 variants induce the formation of a trimeric complex, consisting of pep11, HPV16 E6 and p53. These findings indicate that peptides, which do not contain the LxxLL motif, can reshape E6 to enable its interaction with p53. The formation of the trimeric HPV16 E6 / peptide / p53 complex was associated with an increase of endogenous HPV16 E6 protein amounts. Yet, total cellular p53 amounts were also increased, indicating that the E6 / E6AP-mediated degradation of p53 is blocked. These findings suggest that inhibition of oncogenic activities by targeting the E6AP pocket on HPV16 E6 could be a strategy for therapeutic intervention.

Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.

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.

Oncogenic human papillomaviruses activate the tumor-associated lens epithelial-derived growth factor (LEDGF) gene.

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.

Silencing of human papillomavirus (HPV) E6/E7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from HPV-positive cancer cells.

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.

Small interfering RNAs targeting cyclin D1 and cyclin D2 enhance the cytotoxicity of chemotherapeutic agents in mantle cell lymphoma cell lines.

Cyclin D1 (CCND1) is a known cell cycle regulator whose overexpression is a hallmark of mantle cell lymphoma (MCL). Although molecular techniques have unified the diagnostic approach to MCL, no therapeutic advances have been made to target this particular pathway. The significance of CCND1 in the pathogenesis and treatment of MCL has yet to be defined. We have taken advantage of RNA interference (RNAi) to down-regulate CCND1 expression in two MCL cell lines (Granta-519 and Jeko-1) to investigate the cytotoxic effect of combining RNAi with conventional chemotherapeutic agents. We designed four small interfering RNAs (siRNAs) specific to CCND1, one specific to CCND2, and one dual-targeting siRNA that simultaneously down-regulates CCND1 and CCND2. Etoposide and doxorubicin were used as chemotherapeutics in combination with the siRNAs. The transfected siRNAs in MCL cell lines triggered 40-60% reduction in target mRNA and protein levels. Importantly, the siRNA-mediated reduction in cyclins resulted in decreased IC(50) (50% inhibitory concentration) values for both doxorubicin and etoposide. The combination of siRNA-mediated inhibition of the cyclins along with chemotherapeutic agents could potentially be used to lower the effective doses of the chemotherapeutic agents and reduce drug-related toxicities.

Utilization of AFFX spike-in control probes to monitor sample identity throughout Affymetrix GeneChip Array processing.

Microarrays evolved from a highly specialized technique into a standard molecular biology method that is widely used for whole-genome gene expression profiling. One of the most important aspects of this method is sample identity, that is, whether the expression profile recorded from an array actually derives from the indicated sample. Several potential steps in the protocol exist where a mix-up of samples may occur. With the increasing size of microarray studies, it is important to ensure that each expression profile is assigned to the correct sample. Errors at this level almost certainly lead to erroneous results and can even cause a complete failure of the microarray study. We developed a system that utilizes probes already present on commercially available Affymetrix arrays to unambiguously correlate the recorded expression profile with the input sample RNA. A set of eight spike-in controls were generated, which can be added to sample RNA in different combinations to generate an "on-chip identifier" that passes through the entire array processing protocol and results in a sample-specific hybridization pattern. This pattern can then be used to monitor whether each array was hybridized with the correct sample. The spike-in controls did not have any negative effect on RNA integrity or any detectable influence on the expression values of the remaining probes on the array; therefore, these controls represent an inexpensive and easily adaptable system to guarantee high-quality results from microarray experiments.