Norman Häfner

Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts

Virus integration into the host genome is a characteristic step during cervical carcinogenesis. Experimental data provide evidence that integration could result in increased levels of oncogene (E6/E7) transcripts. This is the first study in which the level of viral transcripts is correlated to the physical state of the viral genome in cervical intraepithelial neoplasia (CIN) and cervical carcinomas (CxCa). Using the APOT-assay integrate-derived transcripts only were detected in 3/28 (11%) CIN and in 28/55 (51%) carcinomas, respectively. The remaining biopsies contained either episome-derived transcripts only or both mRNA species. SybrGreen real time reverse transcriptase–PCR assays were used to quantify viral gene expression for (i) all transcripts initiated from p97, (ii) full-length E6, (iii) E6*I and (iv) E5 transcripts. E6/E7 transcript levels showed a broad distribution but similar median values irrespective of histopathological grading and physical state of the viral genome. Biopsies with integrate-derived transcripts only generally lacked E5-specific mRNA. Our data do not support the hypothesis that HPV integration invariably results in high levels of oncogene transcripts. Instead, constitutive expression of oncogene transcripts rather than the level of expression appears to be decisive for transformation and the maintenance of the malignant phenotype.

HPV16-E6 mRNA is superior to cytokeratin 19 mRNA as a molecular marker for the detection of disseminated tumour cells in sentinel lymph nodes of patients with cervical cancer by quantitative reverse-transcription PCR.

About 10–15% of patients with cervical cancer suffer from recurrence despite histologically negative lymph nodes (pN0). Occult micrometastases or small tumour cell clusters may contribute to disease outcome. The aim of this study was to compare at the RNA level 2 known tumour‐associated genes, HPV16‐E6 and cytokeratin 19 (CK19), as molecular markers for the detection of disseminated tumour cells. Real‐time reverse transcription PCR technology was used to quantify gene expression in histologically positive and negative sentinel lymph nodes (SLN) from 70 patients with cervical cancer. Lymph nodes from noncancer patients were used as controls. Calculated copy numbers were normalised to the geometric average of the most stable housekeeping genes. We observed a good correlation (R = 0.915) between the expression of both markers in SLN with histologically confirmed metastases. However, marker gene expression differed considerably in histologically negative nodes: CK19 transcripts were detected in 90 of 112 SLN (80.4%), whereas only 38 nodes (33.9%) were positive for HPV16 E6 mRNA. In particular, 62 of 74 SLN, which were negative by histology, and HPV16 E6 mRNA expressed CK19 mRNA. Moreover, 8 of 10 lymph nodes from noncancer patients expressed CK19 mRNA. Systematic errors due to RNA degradation or incomplete cDNA could be ruled out. It is concluded that HPV16 E6 mRNA is more specific and more sensitive for the detection of tumour cells in SLN than CK19 mRNA. The specificity of CK19 is limited because of low level expression in uninvolved pelvic lymph nodes.

High expression of crystallin αB represents an independent molecular marker for unfavourable ovarian cancer patient outcome and impairs TRAIL- and cisplatin-induced apoptosis in human ovarian cancer cells

Dysregulated apoptotic pathways are regarded as major reasons for chemoresistance development as a particular challenge in ovarian cancer therapy. In search of molecular factors affecting human ovarian cancer cell apoptosis and, consequently, patient survival, we examined tumors of 103 platinum‐/taxane‐treated ovarian cancer patients by mRNA‐array hybridization, qPCR, and immunohistochemistry. We identified high expression of crystallin αB (CRYAB), a proposed negative regulator of tumor necrosis factor‐related apoptosis inducing ligand (TRAIL)‐mediated apoptosis. By Kaplan Meier analysis, this factor turned out to be significantly associated with poor patient outcome [overall survival (OS) p = 0.001, recurrence‐free survival (RFS) p = 0.003]. Elevated hazard ratios (HR) were estimated with regard to OS (HR = 2.11, 95% CI 1.10–4.06) and RFS (HR = 1.92, 95% CI 1.07–3.47) in multivariable analyses. These associations were confirmed in independent, publicly available mRNA data comprising 431 patients for OS (p < 0.001) and 413 for RFS (p < 0.001). Our findings were validated by studying apoptotic events in cultured human ovarian cancer cells which were stably transfected to express elevated CRYAB levels. These data emphasized the crucial role of CRYAB in human ovarian cancer biology since TRAIL‐ as well as cisplatin‐induced apoptosis was significantly impaired as a function of enhanced CRYAB expression. Taken together, we identified CRYAB as an independent biomarker for unfavourable outcome of human ovarian cancer patients. Since TRAIL is currently tested as anti‐cancer drug and large proportions of the present patient cohort displayed low CRYAB levels in their tumors, CRYAB may enable the selection of patient subgroups benefiting most from TRAIL‐containing therapy.

Hypermethylated DAPK in serum DNA of women with uterine leiomyoma is a biomarker not restricted to cancer

OBJECTIVE Ovarian cancer is most frequently diagnosed at a late stage with a poor prognosis. No markers for early diagnosis have been established. Aberrantly methylated DNA appears as a promising molecular cancer marker. The aim of this study was to analyze the methylation status of the proapoptotic cancer related gene death-associated protein kinase (DAPK) in ovarian cancer patients, healthy controls and in patients suffering from a benign proliferative disease such as uterine leiomyoma. METHODS Methylation-specific PCR (MSP) was used to detect DAPK methylation in primary tumor tissue and serum of both ovarian cancer (n=32) and uterine leiomyoma patients (n=17 primary tissue, n=30 serum). Serum samples from healthy women served as controls (n=20). MSP results were confirmed by restriction digest and sequencing analyses of cloned PCR products. RESULTS DAPK methylation was detected in 50% and 35.3% of primary tissue and 56% and 23.8% of serum samples from ovarian cancer and leiomyoma patients, respectively. However, the association of methylation frequencies in tissue and serum was low (kappa=-0.053). Sequencing experiments revealed fully methylated MSP products in sera of both ovarian cancer and leiomyoma patients. In contrast sera from control patients showed only partially methylated DAPK sequences. CONCLUSION DAPK hypermethylation was neither specific for the tissue of origin nor for cancer. The high prevalence of leiomyoma compromises the utility of this gene as a serum marker for early ovarian cancer detection. These data emphasize the necessity to co-analyze controls presenting with non-cancer proliferative disease in the quest for molecular cancer markers.

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*105 MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC – circulating tumour cells, CxCa – cervical cancer, DD-RT-PCR – Digital-Direct Reverse Transcriptase PCR, HPV – Human Papilloma Virus, MNBC – mononuclear blood cells, ICC – immunocytochemistry.

Novel splice variant CAR 4/6 of the coxsackie adenovirus receptor is differentially expressed in cervical carcinogenesis

The coxsackie adenovirus receptor (CAR) is a component of the tight junction complex and involved in cell adhesion. Loss of CAR expression can affect cell adhesion which in the context of carcinogenesis may influence both invasion and metastatic spread. Functional inactivation of CAR may also result from the interaction with its soluble isoforms. To relate alterations of CAR expression to tumor progression, we aimed to establish a highly specific real-time PCR protocol for quantification of all splice variants. In the process of cloning, we identified a novel splice variant termed CAR4/6 that lacked exon 5 but retained exon 6 encoding the transmembrane domain. Localization of CAR4/6 in the cell membrane was confirmed by ectopic expression in HT1080 cells. Expression analyses using cDNA arrays revealed that most normal tissues, including those of the female genital tract, express full-length CAR (CAR6/7) but not CAR4/6. Differential expression of both CAR splice variants was validated in microdissected epithelia (n = 66) derived from normal cervical ectodermal tissue, high-grade cervical intraepithelial neoplasia (CIN2/3) and invasive squamous cervical carcinoma. CAR4/6 was not expressed in normal cervical tissue but in 42% of CIN2/3 and in most cervical carcinomas (p < 0.001). In contrast, CAR6/7 was detected in all of the microdissected samples. As for CAR4/6 expression levels of CAR6/7 were significantly lower in normal tissue as compared with CIN2/3 and cancer (p < 0.01). Ectopic expression of CAR4/6 in different cell lines enhanced the proliferative and invasive properties indicating a possible role in cancer progression.

An integrative functional genomic and gene expression approach revealed SORBS2 as a putative tumour suppressor gene involved in cervical carcinogenesis

Human papillomavirus (HPV) types 16 and 18 are known to play a major role in cervical carcinogenesis. However, additional genetic alterations are required for the development and progression of cervical cancer. Our aim was to identify genes which are consistently down-regulated in cervical cancers (CxCa) and which are likely to contribute to malignant transformation. Microarray analyses of RNA from high-grade cervical precancers (CIN2/3) and CxCa were performed to screen for putative tumour suppressor genes (TSG) in predefined regions on chromosomes 4 and 10. Validation of the candidate genes was done by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in 16 normal cervical tissues, 14 CIN2/3 and 16 CxCa. The two most promising genes, SORBS2 and CALML5, were expressed ectopically in various cell lines in order to analyse their functional activity. Reconstitution of SORBS2 expression resulted in a significant reduction in cell proliferation, colony formation and anchorage-independent growth in CaSki, HPKII and HaCaT cells, whereby anchorage-independent growth could only be investigated for CaSki cells. SORBS2 had no effect on cell migration. In contrast, reconstitution of CALML5 expression did not influence the phenotype of all cell lines tested. None of the genes could induce senescence or apoptosis. Our results underline a possible role of SORBS2 as a TSG in cervical carcinogenesis.

Platinum(ii) O,S complexes as potential metallodrugs against Cisplatin resistance.

We report on platinum(ii) complexes with different cinnamic acid derivatives as ligands with cytotoxic activity against Cisplatin resistant ovarian cancer cell line subcultures of SKOV3 and A2780. A typical mechanism of action for platinum(ii) complexes as Cisplatin itself is binding to the DNA and inducing double-strand breaks. We examined the biological behavior of these potential drugs with 9-methylguanine using NMR spectroscopic methods and their DNA damage potential including γH2AX-foci analyses. X-ray diffraction methods have been used to elucidate the molecular structures of the platinum(ii) complexes. Interactions with the model protein lysozyme have been evaluated by different techniques including UV-Vis absorption spectroscopy, fluorescence and X-ray crystallography.

TAK1 and IKK2, novel mediators of SCF-induced signaling and potential targets for c-Kit-driven diseases

NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases.

Viral-Cellular DNA Junctions as Molecular Markers for Assessing Intra-Tumor Heterogeneity in Cervical Cancer and for the Detection of Circulating Tumor DNA

The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV) DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA) junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s) were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.