SOX17 expression and its down-regulation by promoter methylation in cervical adenocarcinoma in situ and adenocarcinoma.

AIMS: SOX17 expression has not been studied in glandular lesions of the uterine cervix like adenocarcinoma in situ (AIS) and invasive adenocarcinomas (AdC), whereas SOX17 promoter CpG island methylation has been reported. Therefore, the aim of this study was to relate the topographical distribution of SOX17 expression and SOX17 methylation status to each other, and to SOX2 expression, human papillomavirus (HPV) type, and physical status of the virus. METHODS AND RESULTS: Immunohistochemistry was used in 45 cases to assess expression of SOX17 and SOX2. SOX17 promoter methylation was determined in 25 cases by means of bisulphite conversion and methylation-specific polymerase chain reaction. SOX17 and SOX2 showed a mutually exclusive expression pattern in normal epithelium, with a sharp delineation in the squamocolumnar junction. SOX17 was found in endocervical columnar and reserve cells, whereas SOX2 was exclusively found in squamous epithelium. In both glandular lesions and cases with coexisting glandular and squamous intraepithelial components, a complex combination of SOX17 and SOX2 expression patterns was seen and mutually exclusive expression was lost. Frequently, gain of expression of SOX2 was found and expression of SOX17 was lost. Methylation of the CpG island in the SOX17 promoter was shown to be strongly associated with loss of expression of SOX17 (P = 0.0016). CONCLUSIONS: In this study, we show for the first time a direct correlation between the topographical distribution of SOX17 expression and the methylation status of its gene promoter. This explains the heterogeneity of SOX17 expression in the glandular lesions of the cervix. No correlation was found between HPV type and physical status of the virus on the one hand and methylation status on the other.

Upregulation of AKR1C1 and AKR1C3 expression in OPSCC with integrated HPV16 and HPV-negative tumors is an indicator of poor prognosis.

Different studies have shown that HPV16-positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the levels of viral genes, nor those of virally disrupted human genes, a genome-wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty-three fresh-frozen HPV-16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo-keto-reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV-positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non-hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV-positive (p ≤0.001) and -negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV-negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment.

Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

An exploration of pathways involved in lung carcinoid progression using gene expression profiling.

Pulmonary carcinoids comprise a well-differentiated subset of neuroendocrine tumors usually associated with a favorable prognosis, but mechanisms underlying disease progression are poorly understood. In an explorative approach to identify pathways associated with progression, we compared gene expression profiles of tumors from five patients with a favorable and five with a poor disease outcome. Differentially expressed genes were validated using quantitative real-time PCR on 65 carcinoid tumors, in combination with survival analysis. One of the identified pathways was further examined using immunohistochemistry. As compared with other chromosomal locations, a significantly higher number of genes downregulated in carcinoids with a poor prognosis were located at chromosome 11q (P = 0.00017), a region known to be frequently lost in carcinoids. In addition, a number of upregulated genes were found involved in the mitotic spindle checkpoint, the chromosomal passenger complex (CPC), mitotic kinase CDC2 activity and the BRCA-Fanconi anemia pathway. At the individual gene level, BIRC5 (survivin), BUB1, CD44, IL20RA, KLK12 and OTP were independent predictors of patient outcome. For survivin, the number of positive nuclei was also related to poor prognosis within the group of carcinoids. Aurora B kinase and survivin, major components of the CPC, were particularly upregulated in high-grade carcinomas and may therefore comprise therapeutic targets for these tumors. To our knowledge, this is the first expression profiling study focusing specifically on pulmonary carcinoids and progression. We have identified novel pathways underlying malignant progression and validated several genes as being strong prognostic indicators, some of which could serve as putative therapeutic targets.

Switches of SOX17 and SOX2 expression in the development of squamous metaplasia and squamous intraepithelial lesions of the uterine cervix.

AIMS: The dynamics and topographical distribution of SOX17 and SOX2 expression was studied in the transformation zone (TZ) of the uterine cervix. This TZ is a dynamic area where switches from glandular into squamous epithelium can be recognized, new squamocolumnar junctions are formed, and premalignant lesions originate. SOX17 and SOX2 show mutually exclusive expression patterns in the normal uterine cervix, with SOX2 being exclusively found in squamous epithelium, while SOX17 is detected in endocervical columnar cells and reserve cells. METHODS AND RESULTS: Normal cervices and squamous intraepithelial lesions (SIL) were studied with immunohistochemistry, methylation of SOX17, human papilloma virus (HPV) genotyping, and in situ hybridization. In the TZ squamous metaplasia originating from these reserve cells can still show SOX17 expression, while also remnants of SOX17-positive immature metaplasia can be recognized in the normal squamous epithelium. SOX17 expression is gradually lost during maturation, resulting in the exclusive expression of SOX2 in the majority of (SIL). This loss of SOX17 expression is independent of methylation of the CpG island in its promotor region. HPV can be detected in SOX17-positive immature metaplastic regions in the immediate vicinity of SOX2-positive SIL, suggesting that switches in SOX17 and 2 expression can occur upon HPV infection. CONCLUSIONS: This switch in expression, and the strong association between the distribution of reserve cells and squamous areas within the columnar epithelium in the TZ, suggests that reserve cell proliferations, next to basal cells in the squamous epithelium, are potential targets for the formation of squamous lesions upon viral infection.

The IGF pathway is activated in insulinomas but downregulated in metastatic disease.

Clinical and molecular studies have implicated epidermal growth factor receptor (EGFR), insulin-like growth factor (IGF) and target of rapamycin (mTOR) signaling pathways in the regulation of pancreatic neuroendocrine tumor (PanNET) growth. Interpretation and comparison of these studies is complex due to clinical and molecular tumor heterogeneity. We therefore focused in this study on insulinomas, which we examined for mRNA and protein expression of EGFR, IGF and mTOR signaling pathway components by quantitative real-time PCR (n=48) and immunohistochemistry (n=86). Findings were compared with normal pancreatic islets and correlated with histopathological data and clinical outcome. Insulinomas showed low EGFR and high IGF2 expression. IGFBP2, IGFBP3 and IGFBP6 mRNA levels were 2-4 folds higher than in islets. High protein expression of IGF2, IGF1R and INSR (in 51-92% of the tumors) and low to moderate expression of mTORC1 pathway proteins p-PS6k and p-4EBP1 (7-28% of the tumors) were observed. Correlations were found between 1) ERK1 mRNA expression and that of numerous IGF pathway genes, 2) p-ERK and IGF1R protein expression and 3) decrease of IGF pathway components and both metastatic disease and shorter 10 years disease free survival. In conclusion, our observations suggest that high expression of IGF signaling pathway components is a hallmark of insulinomas, but does not necessarily lead to increased mTOR signaling. Reduced expression of IGF pathway components may be an adverse prognostic factor in insulinomas.

Comprehensive analysis of HPV16 integration in OSCC reveals no significant impact of physical status on viral oncogene and virally disrupted human gene expression.

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16(INK4A) immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

MEN1 gene mutation and reduced expression are associated with poor prognosis in pulmonary carcinoids.

CONTEXT: MEN1 gene alterations have been implicated in lung carcinoids, but their effect on gene expression and disease outcome is unknown. OBJECTIVE: Our objective was to analyze MEN1 gene and expression anomalies in lung neuroendocrine neoplasms and their correlations with clinicopathologic data and disease outcome. DESIGN: We examined 74 lung neuroendocrine neoplasms including 58 carcinoids and 16 high-grade neuroendocrine carcinomas (HGNECs) for MEN1 mutations (n = 70) and allelic losses (n = 69), promoter hypermethylation (n = 65), and mRNA (n = 74) expression. Results were correlated with disease outcome. RESULTS: MEN1 mutations were found in 7 of 55 (13%) carcinoids and in 1 HGNEC, mostly associated with loss of the second allele. MEN1 decreased expression levels correlated with the presence of mutations (P = .0060) and was also lower in HGNECs than carcinoids (P = .0024). MEN1 methylation was not associated with mRNA expression levels. Patients with carcinoids harboring MEN1 mutation and loss had shorter overall survival (P = .039 and P = .035, respectively) and low MEN1 mRNA levels correlated with distant metastasis (P = .00010) and shorter survival (P = .0071). In multivariate analysis, stage and MEN1 allelic loss were independent predictors of prognosis. CONCLUSION: Thirteen percent of pulmonary carcinoids harbor MEN1 mutation associated with reduced mRNA expression and poor prognosis. Also in mutation-negative tumors, low MEN1 gene expression correlates with an adverse disease outcome. Hypermethylation was excluded as the underlying mechanism.

CD44 and OTP are strong prognostic markers for pulmonary carcinoids.

PURPOSE: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histologic classification in typical and atypical carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, that is, orthopedia homeobox (OTP), CD44, and rearranged during transfection (RET). EXPERIMENTAL DESIGN: These genes were analyzed in 56 frozen carcinoids by quantitative real-time PCR (qRT-PCR). RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was conducted on 292 carcinoids. RESULTS: Low mRNA expression levels of CD44 (P = 1.8e(-5)) and OTP (P = 0.00054), and high levels of RET (P = 0.025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP but not for RET. Within all carcinoids as well as atypical carcinoids, absence of CD44 protein was significantly associated with low 20-year survival (P = 0.00014 and 0.00013, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (P = 5.2(-6)). Multivariate analyses revealed that age at diagnosis, histopathology, stage, and cytoplasmic OTP immunoreactivity were independent predictors of prognosis. CONCLUSIONS: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories.

Dual roles of intermediate filaments in apoptosis.

New roles have emerged recently for intermediate filaments (IFs), namely in modulating cell adhesion and growth, and providing resistance to various forms of stress and to apoptosis. In this context, we first summarize findings on the IF association with the cell response to mechanical stress and growth stimulation, in light of growth-related signaling events that are relevant to death-receptor engagement. We then address the molecular mechanisms by which IFs can provide cell resistance to apoptosis initiated by death-receptor stimulation and to necrosis triggered by excessive oxidative stress. In the same way, we examine IF involvement, along with cytolinker participation, in sequential caspase-mediated protein cleavages that are part of the overall cell death execution, particularly those that generate new functional IF protein fragments and uncover neoantigen markers. Finally, we report on the usefulness of these markers as diagnostic tools for disease-related aspects of apoptosis in humans. Clearly, the data accumulated in recent years provide new and significant insights into the multiple functions of IFs, particularly their dual roles in cell response to apoptotic insults.

DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis.

The cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and -9. It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced autocleavage and activation of procaspase-9 in close association with caspase-3.We addressed this issue by investigating DEDD staining patterns in various cell lines and by correlating these expression patterns with the sensitivity of these cell lines for roscovitine-induced apoptosis. We showed that in some cell lines DEDD revealed a bright filamentous staining pattern in others DEDD staining was weak and diffusely distributed in the cytoplasm of the cells. The difference in staining patterns was irrespective of the phosphorylation status of the cytokeratin filaments. In cells showing a filamentous staining pattern, DEDD was strongly associated with the CK8/18 cytokeratin filaments as evidenced by double immunofluorescence and its resistance to extraction with Triton X-100. Subcellular fractionation indicates that DEDD co-purifies with CK18, which corroborates a strong association of DEDD and the cytokeratin network. DEDD was either mono- or diubiquinated. Cells showing a filamentous DEDD distribution are more apoptosis-prone as evidenced by the rapid appearance of M30 CytoDeath-positive cells after induction of apoptosis. The sensitivity towards apoptosis is irrespective of the procaspase-3 content of the cells. Our data support the notion that DEDD-mediated accumulation of procaspases at the cytokeratin scaffold leads to an increase in the local concentration, which renders cells more apoptosis-prone.

Keratin 8/18 breakdown and reorganization during apoptosis.

Monoclonal antibodies that specifically recognize caspase cleaved K18 fragments or specific (phospho)epitopes on intact K8 and K18 were used for a detailed investigation of the temporal and causal relationship of proteolysis and phosphorylation in the collapse of the keratin cytoskeleton during apoptosis. Caspases involved in the specific proteolysis of keratins were analyzed biochemically using recombinant caspases and specific caspase inhibitors. Finally, the fate of the keratin aggregates was analyzed using the M30-ApoptoSense trade mark Elisa kit to measure shedding of caspase cleaved fragments into the supernatant of apoptotic cell cultures. From our studies, we conclude that C-terminal K18 cleavage at the (393)DALD/S site is an early event during apoptosis for which caspase 9 is responsible, both directly and indirectly by activating downstream caspases 3 and 7. Cleavage of the L1-2 linker region of the central alpha-helical rod domain is responsible for the final collapse of the keratin scaffold into large aggregates. Phosphorylation facilitates formation of these aggregates, but is not crucial. K8 and K18 remain associated in heteropolymeric aggregates during apoptosis. At later stages of the apoptotic process, that is, when the integrity of the cytoplasmic membrane becomes compromised, keratin aggregates are shed from the cells.