The impact of cycling hypoxia on the phenotype of HPV-positive cervical cancer cells.

Cycling hypoxia (cycH) is a prevalent form of tumor hypoxia that is characterized by exposure of tumor cells to recurrent phases of hypoxia and reoxygenation. CycH has been associated with a particularly aggressive cellular phenotype of tumor cells and increased therapy resistance. By performing comparative analyses under normoxia, physoxia, chronic hypoxia, and cycH, we here uncover distinct effects of cycH on the phenotype of human papillomavirus (HPV)-positive cervical cancer cells. We show that-other than under chronic hypoxia-viral E6/E7 oncogene expression is largely maintained under cycH as is the E6/E7-dependent regulation of p53 and retinoblastoma protein. Further, cycH enables HPV-positive cancer cells to evade prosenescent chemotherapy, similar to chronic hypoxia. Moreover, cells under cycH exhibit a particularly pronounced resistance to the proapoptotic effects of Cisplatin. Quantitative proteome analyses reveal that cycH induces a unique proteomic signature in cervical cancer cells, which includes a significant downregulation of luminal lysosomal proteins. These encompass the potentially proapoptotic cathepsins B and cathepsin L, which, however, appear not to affect the response to Cisplatin under any of the O2 conditions tested. Rather, we show that the proapoptotic Caspase 8/BH3-interacting domain death agonist (BID) cascade plays a pivotal role for the efficiency of Cisplatin-induced apoptosis in HPV-positive cancer cells under all investigated O2 conditions. In addition, we provide evidence that BID activation by Cisplatin is impaired under cycH, which could contribute to the high resistance to the proapoptotic effects of Cisplatin. Collectively, this study provides the first insights into the profound phenotypic alterations induced by cycH in HPV-positive cancer cells, with implications for their therapeutic susceptibility.

Revisiting the role of endogenous STAT3 in HPV-positive cervical cancer cells.

Novel treatment options for human papillomavirus (HPV)-induced cancers are urgently required. The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is considered to be constitutively active in HPV-positive cervical cancer cells and essential for their proliferation. Moreover, STAT3 was reported to undergo mutually stimulatory interactions with the HPV E6/E7 oncogenes. Thus, inhibiting STAT3 in HPV-positive cancer cells is under discussion to provide a powerful novel therapeutic strategy. We here show that the antifungal drug ciclopirox destabilizes the STAT3 protein by acting as an iron chelator. However, by exploring the functional consequences of STAT3 inhibition in HPV-positive cancer cells, we obtained several unexpected results. Chemical STAT3 inhibitors heterogeneously affect cervical cancer cell proliferation and those which act antiproliferative also block the growth of STAT3 knockout cells, indicating induction of off-target effects. In contrast to several chemical inhibitors, genetic inhibition of STAT3 expression by either RNA interference or the CRISPR/Cas9 method does not appreciably affect cervical cancer cell proliferation. Transcriptome analyses indicate that blocking STAT3 expression in HPV-positive cancer cells has very limited effects on putative STAT3 target genes. Although the targeted inhibition of specific growth-promoting signaling pathways leads to a feedback activation of STAT3 in cervical cancer cells via Janus kinase 1/2, this does not lead to treatment resistance. Moreover, we did not obtain experimental evidence for a STAT3-linked activation of HPV E6/E7 oncogene expression or, vice versa, an E6/E7-dependent activation of STAT3, at endogenous conditions in cervical cancer cells. Collectively, these findings question the essential role of STAT3 in cervical cancer cell proliferation and the strategy to inhibit STAT3 in these cells for therapeutic purposes.

Dickkopf-1 expression is repressed by oncogenic human papillomaviruses (HPVs) and regulates the Cisplatin sensitivity of HPV-positive cancer cells in a JNK-dependent manner.

Oncogenic human papillomavirus (HPV) types control the phenotype of cervical cancer cells through the sustained expression of the viral E6/E7 oncogenes. Here, we show that they strongly restrain expression of the putative tumor suppressor protein Dkk1 (Dickkopf-1) in HPV-positive cervical cancer cells through the restriction of p53 expression by the continuously expressed endogenous E6 oncoprotein. Moreover, our study reveals that compromised Dkk1 expression is linked to increased resistance of HPV-positive cervical cancer cells toward the proapoptotic activity of Cisplatin. Although Dkk1 can act as a Wnt antagonist, the antiapoptotic effect resulting from Dkk1 repression is not linked to an activation of this pathway. Rather, transcriptome and functional analyses uncover that Dkk1 repression leads to a strongly diminished stimulation of c-Jun N-terminal kinase (JNK) signaling which is required for efficient apoptosis induction by Cisplatin in cervical cancer cells. Further, we observed that Dkk1-depleted cervical cancer cells induce senescence under Cisplatin treatment instead of apoptosis, suggesting that Dkk1 levels can strongly influence the phenotypic response of these cells toward Cisplatin. Collectively, these results provide new insights into the virus/host cell crosstalk in cervical cancer cells by identifying Dkk1 as a cellular target which is maintained under strong negative control by the continuous expression of the HPV oncogenes. Moreover, they identify Dkk1 as a critical determinant for the sensitivity of cervical cancer cells toward Cisplatin, showing that Dkk1 repression leads to increased Cisplatin resistance by impairing proapoptotic JNK signaling.

Effects of Metformin on the virus/host cell crosstalk in human papillomavirus-positive cancer cells.

Oncogenic types of human papillomaviruses (HPVs) are major human carcinogens. The viral E6/E7 oncogenes maintain the malignant growth of HPV-positive cancer cells. Targeted E6/E7 inhibition results in efficient induction of cellular senescence, which could be exploited for therapeutic purposes. Here we show that viral E6/E7 expression is strongly downregulated by Metformin in HPV-positive cervical cancer and head and neck cancer cells, both at the transcript and protein level. Metformin-induced E6/E7 repression is glucose and PI3K-dependent but-other than E6/E7 repression under hypoxia-AKT-independent. Proteome analyses reveal that Metformin-induced HPV oncogene repression is linked to the downregulation of cellular factors associated with E6/E7 expression in HPV-positive cancer biopsies. Notably, despite efficient E6/E7 repression, Metformin induces only a reversible proliferative stop in HPV-positive cancer cells and enables them to evade senescence. Metformin also efficiently blocks senescence induction in HPV-positive cancer cells in response to targeted E6/E7 inhibition by RNA interference. Moreover, Metformin treatment enables HPV-positive cancer cells to escape from chemotherapy-induced senescence. These findings uncover profound effects of Metformin on the virus/host cell interactions and the phenotype of HPV-positive cancer cells with implications for therapy-induced senescence, for attempts to repurpose Metformin as an anticancer agent and for the development of E6/E7-inhibitory therapeutic strategies.

Effects of the antifungal agent ciclopirox in HPV-positive cancer cells: Repression of viral E6/E7 oncogene expression and induction of senescence and apoptosis.

The malignant growth of human papillomavirus (HPV)-positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV-positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV-positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of prosenescent agents, CPX-induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53-independent Caspase-3/7 activation and induction of apoptosis. CPX also eliminates HPV-positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound antiviral and antiproliferative effects in HPV-positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV-inhibitory, prosenescent and proapoptotic agents in both normoxic and hypoxic environments.

Induction of dormancy in hypoxic human papillomavirus-positive cancer cells.

Oncogenic human papillomaviruses (HPVs) are closely linked to major human malignancies, including cervical and head and neck cancers. It is widely assumed that HPV-positive cancer cells are under selection pressure to continuously express the viral E6/E7 oncogenes, that their intracellular p53 levels are reconstituted on E6/E7 repression, and that E6/E7 inhibition phenotypically results in cellular senescence. Here we show that hypoxic conditions, as are often found in subregions of cervical and head and neck cancers, enable HPV-positive cancer cells to escape from these regulatory principles: E6/E7 is efficiently repressed, yet, p53 levels do not increase. Moreover, E6/E7 repression under hypoxia does not result in cellular senescence, owing to hypoxia-associated impaired mechanistic target of rapamycin (mTOR) signaling via the inhibitory REDD1/TSC2 axis. Instead, a reversible growth arrest is induced that can be overcome by reoxygenation. Impairment of mTOR signaling also interfered with the senescence response of hypoxic HPV-positive cancer cells toward prosenescent chemotherapy in vitro. Collectively, these findings indicate that hypoxic HPV-positive cancer cells can induce a reversible state of dormancy, with decreased viral antigen synthesis and increased therapeutic resistance, and may serve as reservoirs for tumor recurrence on reoxygenation.

PI3K/AKT/mTOR Signaling Regulates the Virus/Host Cell Crosstalk in HPV-Positive Cervical Cancer Cells.

Human papillomavirus (HPV)-induced cancers will remain a significant clinical challenge for decades. Thus, the development of novel treatment strategies is urgently required, which should benefit from improving our understanding of the mechanisms of HPV-induced cell transformation. This should also include analyses of hypoxic tumor cells, which represent a major problem for cancer therapy. Recent evidence indicates that the PI3K/AKT/mTOR network plays a key role for the virus/host cell crosstalk in both normoxic and hypoxic HPV-positive cancer cells. In normoxic cells, the efficacy of the senescence induction upon experimental E6/E7 repression depends on active mTORC1 signaling. Under hypoxia, however, HPV-positive cancer cells can evade senescence due to hypoxic impairment of mTORC1 signaling, albeit the cells strongly downregulate E6/E7. Hypoxic repression of E6/E7 is mediated by the AKT kinase, which is activated under hypoxia by its canonical upstream regulators mTORC2 and PI3K. This review highlights our current knowledge about the oxygen-dependent crosstalk of the PI3K/AKT/mTOR signaling circuit with the HPV oncogenes and the phenotypic state of the host cell. Moreover, since the PI3K/AKT/mTOR pathway is considered to be a promising target for anticancer therapy, we discuss clinical implications for the treatment of HPV-positive cervical and head and neck squamous cell carcinomas.

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.

The inhibitors of nucleotide biosynthesis leflunomide, FK778, and mycophenolic acid activate hepatitis B virus replication in vitro.

UNLABELLED: The inhibitors of pyrimidine synthesis, leflunomide and FK778, have been reported to exert broad antiviral effects, in addition to their immunosuppressive activities. Their possible therapeutic benefit for transplantation medicine is currently discussed, because they also block the replication of human cytomegalovirus and human polyomavirus BK, which both cause important complications in transplant recipients. Here, we show that leflunomide and FK778 strongly enhance hepatitis B virus (HBV) replication in vitro. This activity is shared by mycophenolic acid (MPA), an inhibitor of purine biosynthesis. Stimulation of HBV replication by these agents was linked to their inhibitory effects on de novo nucleotide biosynthesis because it could be efficiently counteracted by external nucleoside supply. Mechanistically, we found that mitogen-activated protein kinase p38 played a key role for the enhancement of HBV replication by leflunomide, FK778, and MPA. All three HBV-activating compounds increased p38 phosphorylation, in contrast to the HBV inhibitors, telbivudine and cyclosporine A. Moreover, silencing of p38 expression through RNA interference efficiently counteracted the stimulatory effect of leflunomide, FK778, and MPA on HBV replication. CONCLUSION: Our data indicate that, in contrast to their reported inhibitory effects on other viruses, both leflunomide and FK778 can augment HBV replication. Treatment with leflunomide, FK778, or MPA may bear the risk to enhance HBV replication in infected patients.

FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.

The FAM57A (family with sequence similarity 57 member A) gene is controversially discussed to possess pro- or anti-tumorigenic potential. Here, we analyze the regulation of cellular FAM57A protein levels and study the functional role of FAM57A in HPV-positive cervical cancer cells. We find that FAM57A protein expression strongly depends on cell density, with FAM57A being readily detectable at low cell density, but undetectable at high cell density. This regulation occurs post-transcriptionally and is not mirrored by corresponding changes at the RNA level. We further show that FAM57A protein levels are highly increased in cervical cancer cells cultivated at hypoxia compared to normoxia and provide evidence that FAM57A is a hypoxia-responsive gene under control of the α-subunit of the HIF-1 (hypoxia-inducible factor-1) transcription factor. Yet, the strong relative increase of FAM57A protein levels in hypoxic cells is predominantly cell-density-dependent and occurs post-transcriptionally. Other anti-proliferative effectors besides hypoxia, such as silencing of HPV E6/E7 oncogene expression in cervical cancer cells, also result in an increase of FAM57A levels compared to untreated cells. Functional analyses reveal that FAM57A repression leads to pronounced anti-proliferative as well as anti-migratory effects in cervical cancer cells. Taken together, these results provide insights into the regulation of FAM57A protein levels and reveal that they underlie a tight cell-density-dependent control. Moreover, they identify FAM57A as a critical determinant for the phenotype of cervical cancer cells, which promotes their proliferation and migration capacities.

Emerging topics in human tumor virology.

After a long period of scepticism and disbelief, tumor viruses are today recognized as a significant cancer risk factor for humans. Much has been learned about the viral transforming mechanisms and prophylactic vaccines have been developed against 2 major tumor viruses, HBV and HPV. Yet, many important issues of tumor virology remain unresolved and exciting new ones are emerging from recent discoveries. They define future research directions for the field and include (i) novel strategies for tumor virus hunting, (ii) tumor viruses as experimental tools to study human carcinogenesis, (iii) the interplay between viruses and the world of small noncoding RNAs, (iv) epigenetic interactions between tumor viruses and the host cell, (v) the role of virus/virus interactions for viral carcinogenesis and (vi) novel strategies for prevention and therapy of virus-associated cancers. These topics are discussed by summarizing recent developments, pointing out unresolved issues and suggesting possible strategies for their solution.

Isolation of peptides blocking the function of anti-apoptotic Livin protein.

Livin (ML-IAP) is a cancer-associated member of the inhibitor of apoptosis protein (IAP) family. By yeast two-hybrid screening of a randomized peptide expression library, we isolated short linear peptides that specifically bind to Livin, but not to other IAPs. Intracellular expression of the peptides sensitized livin-expressing cancer cells toward different pro-apoptotic stimuli. The bioactive peptides neither showed sequence homologies to Smac-derived IAP inhibitors, nor did they interfere with the binding of Livin to Smac. Intracellular expression of the peptides did not affect the levels or the subcellular distribution of Livin. Growth of livin-expressing tumor cells was inhibited in colony formation assays by the Livin-targeting peptides. These findings provide evidence that the targeted inhibition of Livin by peptides represents a viable approach for the apoptotic sensitization and growth inhibition of tumor cells. The inhibitory peptides isolated here could form a novel basis for the development of therapeutically useful Livin inhibitors.

Delineating the Switch between Senescence and Apoptosis in Cervical Cancer Cells under Ciclopirox Treatment.

The iron-chelating drug ciclopirox (CPX) may possess therapeutic potential for cancer treatment, including cervical cancer. As is observed for other chemotherapeutic drugs, CPX can induce senescence or apoptosis in cervical cancer cells which could differently affect their therapy response. The present study aims to gain insights into the determinants which govern the switch between senescence and apoptosis in cervical cancer cells. We performed proteome analyses, proliferation studies by live-cell imaging and colony formation assays, senescence and apoptosis assays, and combination treatments of CPX with inhibitors of oxidative phosphorylation (OXPHOS) or glycolysis. We found that CPX downregulates OXPHOS factors and facilitates the induction of apoptosis under limited glucose availability, an effect which is shared by classical OXPHOS inhibitors. Under increased glucose availability, however, CPX-induced apoptosis is prevented and senescence is induced, an activity which is not exerted by classical OXPHOS inhibitors, but by other iron chelators. Moreover, we show that the combination of CPX with glycolysis inhibitors blocks cervical cancer proliferation in a synergistic manner. Collectively, our results reveal that the phenotypic response of cervical cancer cells towards CPX is strongly dependent on glucose availability, link the pro-apoptotic and pro-senescent activities of CPX to its bifunctionality as an OXPHOS inhibitor and iron chelator, respectively, and provide a rationale for combining CPX with glycolysis inhibitors.

ITIH5 shows tumor suppressive properties in cervical cancer cells grown as multicellular tumor spheroids.

Cervical cancer (CC) arises from premalignant cervical intraepithelial neoplasia (CIN) induced by a persistent infection with human papillomaviruses. The multi-stepwise disease progression is driven by genetic and epigenetic alterations. Our previous studies demonstrated a clear downregulation of inter-α-trypsin-inhibitor-heavy chain 5 (ITIH5) at mRNA and protein levels in CC compared to CIN2/3 and normal cervical tissue. Initial in vitro functional analyses revealed a suppressive effect of ITIH5 on relevant mechanisms for cancer progression in conventional two dimensional (2D) cell culture model systems. Based on these studies, we aimed to investigate the functional relevance of ITIH5 in multicellular tumor spheroid (MCTS) models, which resemble in vivo tumors more closely. We successfully established CC cell line-derived MCTS using the hanging-drop technique. ITIH5 was ectopically overexpressed in HeLa and SiHa cells and its functional relevance was investigated under three dimensional (3D) culture conditions. We found that ITIH5 re-expression significantly suppressed tumor spheroid growth and spheroid invasiveness of both HeLa and SiHa spheroids. Immunohistochemical (IHC) analyses revealed a significant reduction in Ki-67 cell proliferation index and CAIX-positive areas indicative for hypoxia and acidification. Furthermore, we observed an increase in cPARP-positive cells suggesting a higher rate of apoptosis upon ITIH5 overexpression. An effect of ITIH5 expression on the susceptibility of cervical MCTS towards cytostatic drug treatment was not observed. Collectively, these data uncover pronounced anti-proliferative effects of ITIH5 under 3D cell culture conditions and provide further functional evidence that the downregulation of ITIH5 expression during cervical carcinogenesis could support cancer development.

Transtactin: a universal transmembrane delivery system for Strep-tag II-fused cargos.

The delivery of molecules into cells poses a critical problem that has to be solved for the development of diagnostic tools and therapeutic agents acting on intracellular targets. Cargos which by themselves cannot penetrate cellular membranes due to their biophysical properties can achieve cell membrane permeability by fusion to protein transduction domains (PTDs). Here, we engineered a universal delivery system based on PTD-fused Strep-Tactin, which we named Transtactin. Biochemical characterization of Transtactin variants bearing different PTDs indicated high thermal stabilities and robust secondary structures. Internalization studies demonstrated that Transtactins facilitated simple and safe transport of Strep-tag II-linked small molecules, peptides and multicomponent complexes, or biotinylated proteins into cultured human cells. Transtactin-introduced cargos were functionally active, as shown for horseradish peroxidase serving as a model protein. Our results demonstrate that Transtactin provides a universal and efficient delivery system for Strep-tag II-fused cargos.

Targeting Id1 and Id3 by a specific peptide aptamer induces E-box promoter activity, cell cycle arrest, and apoptosis in breast cancer cells.

Inhibitors of differentiation or DNA binding (Id) proteins have been shown to be involved in tumor growth, invasiveness, metastasis, and angiogenesis. Overexpression of Id proteins, especially Id1, correlates with unfavorable clinical prognosis. Thus, they are attractive molecular targets for anticancer therapy. Overexpression of Id proteins mediates breast cancer metastasis to lung. Targeting Id1 and Id3 expression in breast cancer cells reduces breast cancer metastasis in animal models. Different breast tumors failed to grow and/or metastasize in Id1 (+/-) Id3 (-/-) mice. Id1 and Id3 preferentially dimerize with the key regulatory E-proteins which inhibit the expression of different tumor suppressor genes. Nevertheless, the inhibition of tumorigenic activities of Id1 and Id3 at protein level has never been studied. Here, we isolated a novel peptide aptamer, Id1/3-PA7, specifically interacting with Id1 and Id3 from randomized combinatorial expression library using yeast and mammalian two-hybrid systems. Intracellular delivered Id1/3-PA7 co-localized to Id1 and Id3 and interfered with their functions. It repressed E47 protein sequestration by Id1 and Id3, activated the E-box promoter and increased the expression level of cyclin-dependent kinase inhibitors (CDKN1A and CDKN1B) in a dose-dependent fashion, paralleled by the cleavage of poly ADP ribose polymerase (PARP). These effects were counteracted by ectopically overexpressed Id1 and Id3. Peptide aptamer Id1/3-PA7 induced cell cycle arrest and apoptosis in breast cancer cells MCF7 and MDA-MB-231. In conclusion, Id1/3-PA7 could represent a nontoxic exogenous agent that can significantly provoke antiproliferative and apoptotic effects in breast cancer cells, which are associated with deregulated expression of Id1 and Id3.

Enhancer of zeste homolog 2 (EZH2) expression is an independent prognostic factor in renal cell carcinoma.

BACKGROUND: The enhancer of zeste homolog 2 (EZH2) gene exerts oncogene-like activities and its (over)expression has been linked to several human malignancies. Here, we studied a possible association between EZH2 expression and prognosis in patients with renal cell carcinoma (RCC). METHODS: EZH2 protein expression in RCC specimens was analyzed by immunohistochemistry using a tissue microarray (TMA) containing RCC tumor tissue and corresponding normal tissue samples of 520 patients. For immunohistochemical assessment of EZH2 expression, nuclear staining quantity was evaluated using a semiquantitative score. The effect of EZH2 expression on cancer specific survival (CSS) was assessed by univariate and multivariate Cox regression analyses. RESULTS: During follow-up, 147 patients (28%) had died of their disease, median follow-up of patients still alive was 6.0 years (range 0-16.1 years). EZH2 nuclear staining was present in tumor cores of 411 (79%) patients. A multivariate Cox regression analysis revealed that high nuclear EZH2 expression was an independent predictor of poor CSS (> 25-50% vs. 0%: HR 2.72, p = 0.025) in patients suffering from non-metastatic RCC. Apart from high nuclear EZH2 expression, tumor stage and Fuhrman's grading emerged as significant prognostic markers. In metastatic disease, nuclear EZH2 expression and histopathological subtype were independent predictive parameters of poor CSS (EZH2: 1-5%: HR 2.63, p = 0.043, >5-25%: HR 3.35, p = 0.013, >25%-50%: HR 4.92, p = 0.003, all compared to 0%: HR 0.36, p = 0.025, respectively). CONCLUSIONS: This study defines EZH2 as a powerful independent unfavourable prognostic marker of CSS in patients with metastatic and non-metastatic RCC.

Membrane partitioning of peptide aggregates: coarse-grained molecular dynamics simulations.

Coarse-grained molecular dynamics (CGMD) simulation technique (MARTINI force field) is applied to monitor the aggregation of helical peptides representing the transmembrane sequence and its extension of bone marrow stromal cell antigen 2 (BST-2). One of the peptides is coupled with a protein transducing domain (PTD) of nine arginine residues (R9) at its N-terminal side as well as a peptide, pep11**, which has been shown to bind to human papilloma virus 16 (HPV16) E6 oncoprotein. A short hydrophobic stretch of the transmembrane domain (TMD) of BST-2 aggregates the fastest and inserts into a lipid membrane. An aggregate of R9-pep11** attaches to the membrane via simultaneous contact of many arginine residues. Monomers from the aggregates of the shortest of the hydrophobic TMDs dissolve into the opposing leaflet when the aggregate spans the bilayer. A 'flipping' of the individual monomeric peptides is not observed.Communicated by Ramaswamy H. Sarma.