Sphingosine-kinase-1 expression is associated with improved overall survival in high-grade serous ovarian cancer.

PURPOSE: Sphingosine-kinase-1 (SPHK1) is a key enzyme of sphingolipid metabolism which is involved in ovarian cancer pathogenesis, progression and mechanisms of drug resistance. It is overexpressed in a variety of cancer subtypes. We investigated SPHK1 expression as a prognostic factor in epithelial ovarian cancer patients. METHODS: Expression analysis of SPHK1 was performed on formalin-fixed paraffin-embedded tissue from 1005 ovarian cancer patients with different histological subtypes using immunohistochemistry. Staining intensity of positive tumor cells was assessed semi-quantitatively, and results were correlated with clinicopathological characteristics and survival. RESULTS: In our ovarian cancer collective, high levels of SPHK1 expression correlated significantly with complete surgical tumor resection (p = 0.002) and lower FIGO stage (p = 0.04). Progression-free and overall survival were further significantly longer in patients with high-grade serous ovarian cancer and overexpression of SPHK1 (p = 0.002 and p = 0.006, respectively). CONCLUSION: Our data identify high levels of SPHK1 expression as a potential favorable prognostic marker in ovarian cancer patients.

Tumor mutational burden and immune infiltration as independent predictors of response to neoadjuvant immune checkpoint inhibition in early TNBC in GeparNuevo.

BACKGROUND: The predictive value of tumor mutational burden (TMB), alone or in combination with an immune gene expression profile (GEP), for response to neoadjuvant therapy in early triple negative breast cancer (TNBC) is currently not known, either for immune checkpoint blockade (ICB) or conventional chemotherapy. PATIENTS AND METHODS: We obtained both whole exome sequencing and RNA-Seq data from pretreatment samples of 149 TNBC of the recent neoadjuvant ICB trial, GeparNuevo. In a predefined analysis, we assessed the predictive value of TMB and a previously developed immune GEP for pathological complete remission (pCR). RESULTS: Median TMB was 1.52 mut/Mb (range 0.02-7.65) and was significantly higher in patients with pCR (median 1.87 versus 1.39; P = 0.005). In multivariate analysis, odds ratios for pCR per mut/Mb were 2.06 [95% confidence intervals (CI) 1.33-3.20, P = 0.001] among all patients, 1.77 (95% CI 1.00-3.13, P = 0.049) in the durvalumab treatment arm, and 2.82 (95% CI 1.21-6.54, P = 0.016) in the placebo treatment arm, respectively. We also found that both continuous TMB and immune GEP (or tumor infiltrating lymphocytes) independently predicted pCR. When we stratified patients in groups based on the upper tertile of TMB and median GEP, we observed a pCR rate of 82% (95% CI 60% to 95%) in the group with both high TMB and GEP in contrast to only 28% (95% CI 16% to 43%) in the group with both low TMB and GEP. CONCLUSIONS: TMB and immune GEP add independent value for pCR prediction. Our results recommend further analysis of TMB in combination with immune parameters to individually tailor therapies in breast cancer.

Molecular Markers as Prognostic Factors in DCIS and Small Invasive Breast Cancers.

Ductal carcinoma in situ (DCIS) accounts for up to half of screen-detected breast cancers and thus constitutes a major public health problem. Despite effective current treatment many patients with DCIS are either over- or undertreated because of the paucity of precise models to predict recurrence or progression. The combination of clinical and molecular factors as already applied for invasive disease may help to build such models also for DCIS. We compared 53 DCIS (36.6 %) and 92 (63.4 %) invasive breast cancer cases and found no significant differences in age, receptor status of ER, PR, and HER2, and the use of radiotherapy. Interestingly, the proportion of disseminated tumor cells (DTC) did also not significantly differ between DCIS and invasive cases (p = 0.57). A negative PR status was associated with the detection of DTCs (p = 0.026). We then compared relationships of clinical parameters and biomarkers with patients' prognosis in 43 DCIS and 40 small invasive tumors ≤ 5 mm (T1a). ER negativity was associated with shorter relapse free survival in the complete cohort (p = 0.004) and showed a trend in both subgroups (p = 0.053 for DCIS and p = 0.046 for T1a, respectively). In conclusion, we found markedly similar properties of both DCIS and small invasive breast cancers with respect to the distribution of several parameters as well as to the prognostic value of biomarkers. DCIS with a luminal phenotype seem to be characterized by a favourable prognosis.

Prognostic impact of thymidine phosphorylase expression in breast cancer--comparison of microarray and immunohistochemical data.

Contrary findings exist according to the prognostic and predictive impact of thymidine phosphorylase (TP) expression in breast cancer. Goal of our study was to investigate TP expression on the mRNA level by microarray analysis in a large cohort of 1781 breast cancers and to analyse its prognostic impact. Furthermore we compared mRNA expression and immunohistochemical data to explain discrepancies between different studies. The prognostic value of TP mRNA expression was analysed among n=622 untreated patients. Strong expression in the subgroup of n=213 ER-negative cancer correlates with improved survival (P=0.012). In contrast, no difference in survival was detected in the ER-positive group. We also failed to observe a prognostic value of TP mRNA among n=435 endocrine-treated patients as well as n=111 CMF-treated patients. In an unsupervised analysis, TP clustered together with genes expressed in immune cells. Moreover, among normal tissues the highest TP mRNA expression was found in tissues of the immune system. The profile of TP expression in breast cancers correlates to a metagene of interferon induction whereas the expression of TP among normal tissues correlates to a metagene for macrophages. When comparing microarray data with immunohistochemistry from the same n=51 samples, there was no correlation with stained carcinoma cells. In contrast, the correlation with stromal staining was highly significant (P<0.001). Thus TP mRNA from microarray mainly reflects expression in stromal and immune cells. This could account for discrepant results from mRNA and IHC studies. In conclusion, the tumour infiltrating immune cells seem to be a major source of TP expression and predict a favourable prognosis in ER-negative breast cancer. Our data point to a role of TP in host immune response.

Clinical relevance of the putative stem cell marker p63 in breast cancer.

P63 is a member of the p53 family. This protein is crucial for the maintenance of a stem cell population in the human epithelium and necessary for the normal development of all epithelial tissues including mammary glands. In normal breast tissue, the p63 seems to be a specific myoepithelial cell marker. P63 expression has been described in highly aggressive ER negative basal-like breast tumors. The value of p63 expression in ER positive disease is less clear. The expression levels of p63 mRNA by Affymetrix microarray analysis in a combined cohort of 2,158 ER positive breast cancers and its prognostic and predictive impact were analyzed. Tumor samples containing large amounts of benign breast tissue, which will interfere with p63 measurement, were excluded prior to the analysis. Survival analysis revealed a better prognosis of ER positive breast cancer expressing p63 (n = 410; P < 0.036). No correlation of p63 with standard parameters was observed. In a subgroup analysis, endocrine-treated patients with high p63 expression showed a better prognosis than low p63 expression (P = 0.06; n = 186). In untreated patients, this effect was less clear (n = 148; P = 0.5). P63 is a positive prognostic factor in endocrine-treated ER positive breast cancer and might influence responsiveness to endocrine treatment. Thus, p63 could be helpful as a predictive factor for endocrine therapy.

Acid ceramidase 1 expression correlates with a better prognosis in ER-positive breast cancer.

OBJECTIVES: Ceramide and sphingosine mediate response to cancer therapy, inhibit cell growth and induce apoptosis in vitro. Only a few clinical data about the impact of ceramide and sphingosine iny vivo are available. We investigated the relevance of ceramide- and sphingosine-generating enzymes in breast cancer (acid ceramidase 1 (ASAH1), ceramide synthases 4 (LASS4) and 6 (LASS6)) by means of gene expression analysis. METHODS: We analyzed differences in ASAH1, LASS4 and LASS6 on mRNA level between breast cancer subgroups using microarray data from 1581 tumor samples. RESULTS: High ASAH1, LASS4 and LASS6 expression correlates with pathohistological grading (p < 0.001) and estrogen receptor (ER) status (p < 0.001). High ASAH1 expression was associated with a larger tumor size >2 cm (p = 0.003), while high LASS6 expression was correlated with ErbB2 negativity (p < 0.001). In survival analysis, we detected a significant better prognosis of patients with higher ASAH1 expression (p = 0.002) in the ER-positive subgroup. In contrast, expression of LASS4 or LASS6 did not show any prognostic impact. In the multivariate analysis, only ASAH1 expression (p = 0.002), tumor size (p < 0.0001) and ErbB2 positivity (p = 0.041) remained significant. CONCLUSION: ASAH1 is an estrogen-dependent member of the sphingolipid metabolism, which might provide further prognostic information in ER-positive breast cancers.

Gene expression of topoisomerase II alpha (TOP2A) by microarray analysis is highly prognostic in estrogen receptor (ER) positive breast cancer.

INTRODUCTION: Overexpression of Topoisomerase II alpha (TOP2A) has been implicated with gene amplification of the 17q21 amplicon and consecutively with ErbB2 overexpression and amplification. However, gene amplification does not necessarily correlate with RNA and protein expression. There is growing evidence that TOP2A protein expression is a strong prognostic and TOP2A gene amplification might be a predictive marker (particularly for the use of anthracyclines). METHODS: Large scale analysis was performed using Affymetrix microarray data from n = 1,681 breast cancer patients to evaluate TOP2A expression. RESULTS: TOP2A expression showed a strong correlation with tumor size (chi(2)-test, P < 0.001), grading (P < 0.001), ErbB2 (P < 0.001) and Ki67 expression (P < 0.001) as well as nodal status (P = 0.042). Survival analysis revealed a significant prognostic value in ER positive (n = 994; log rank P < 0.001), but not in ER negative breast cancer patients (n = 369, P = 0.35). The prognostic impact of TOP2A expression was independent of Ki67 expression in ER positive tumors (P = 0.002 and P = 0.007 for high and low Ki67, respectively). Moreover a worse prognosis of high TOP2A expressing tumors was found in the subgroup of ErbB2 negative tumors (P < 0.001) and a trend among ErbB2 positive tumors (P = 0.11). The prognostic value of TOP2A was independent of whether the patients were untreated or had received adjuvant therapy. In multivariate Cox regression analysis including standard parameters TOP2A emerged to be the top prognostic marker (HR 2.40, 95% CI 1.68-3.43, P < 0.001). CONCLUSION: TOP2A expression is an independent prognostic factor in ER positive breast cancer and could be helpful for risk assessment in ER positive breast cancer patients.

Differentially expressed genes of reprogrammed human pluripotent stem cells in breast cancer.

Reprogramming of human somatic cells into pluripotent cell types gives insight in the pathophysiology of diseases. We analysed genes recently shown to be differentially expressed in induced pluripotent stem cells (iPS) in 95 breast cancer samples. This analysis reveals two breast cancer subgroups with stem cell-like features, differing in ER-status and proliferation as well as in their clinical course of disease.

"Stem cell like" breast cancers-a model for the identification of new prognostic/predictive markers in endocrine responsive breast cancer exemplified by Plexin B1.

The identification of new biological markers for breast cancer has adopted a new dimension by the use of novel techniques such as global gene expression profiling. While important results have been achieved by these methods not all hopes for a more precise assessment of patients' prognosis have yet been accomplished and validation of prognostic or predictive gene signatures is still often difficult. Several recent approaches suggest that comparisons of differential gene expression could be more instructive if prior classifications of tumors based on molecular or biological characteristics were applied. We previously reported a subtype of breast cancer by using a cluster of coordinately expressed genes many of which has been associated with the mammary epithelial stem cells. While a stringent inverse link of ER status and proliferation of the tumor was observed among those "stem cell like" (SCL) tumors, this link was "uncoupled" in about half of the Non-"stem cell like" (Non-SCL) tumors. This subgroup of SCL tumors can be used as a reference system to analyze changes in the ER pathway by comparing the expression of genes dependent on the ER status. By using this strategy we identified Plexin B1, a cell-surface receptor for the semaphorin Sema4D, whose expression is reduced in the group of "uncoupled" tumors. Loss of Plexin B1 is associated with a poor prognosis in both univariate (all patients: p=0.0062; ER positive: p=0.0107) and multivariate analyses (all patients: p=0.032; ER positive: p=0.022). In conclusion those strategies of gene expression analysis in a context of biological meaningful classifications could be helpful to reveal new prognostic/predictive markers.

The erbB2+ cluster of the intrinsic gene set predicts tumor response of breast cancer patients receiving neoadjuvant chemotherapy with docetaxel, doxorubicin and cyclophosphamide within the GEPARTRIO trial.

Gene expression profiling using Affymetrix HG-U133 Arrays (22,500 genes) was performed on fresh frozen pretherapeutic core biopsies from 50 patients undergoing neoadjuvant chemotherapy (NAC) with docetaxel, adriamycin, cyclophosphamide (TAC) within the GEPARTRIO trial. The Sorlie classification based on the "intrinsic gene set" revealed four different subgroups in our cohort (normal-like: 14%, basal-like: 20%, erbB2+: 22% and luminal: 44%), which is in line with the original description. High genomic grade but not histopathological grading was statistically different within the four subgroups (P<0.001). About 45.5% of tumors classified according to erbB2+ cluster showed a pathological complete response compared to 0% in the normal-like, 10.0% in the basal-like and 9.1% in the luminal subgroup (P=0.024). There was a trend to less tumor relapses in the erbB2+ subgroup (0%) compared to the normal-like (28.6%), basal-like (30.0%) and luminal (13.6%) cluster (P=0.215). Our data suggest that the molecular tumor subtypes based on the "intrinsic gene set" can be used to predict tumor response according to NAC.

Gene expression profiling of breast cancer patients treated with docetaxel, doxorubicin, and cyclophosphamide within the GEPARTRIO trial: HER-2, but not topoisomerase II alpha and microtubule-associated protein tau, is highly predictive of tumor response.

Gene expression analysis in breast cancer patients undergoing neoadjuvant chemotherapy is an interesting tool for identification of gene signatures and new markers to predict tumor response. However, the detection of predictive markers strongly depends on the drugs used in the specific therapeutic setting. There is growing evidence that topoisomerase II-alpha (TOPO IIalpha) is a marker for anthracycline-, and microtubule-associated protein tau (MAPT) for taxane sensitivity. HER-2 has been described as a marker of both anthracycline and taxane sensitivity. We performed gene expression profiling of 50 patients within the GEPARTRIO study, an anthracycline and taxane neoadjuvant chemotherapy trial. Here we investigate the predictive value of TOPO IIalpha, MAPT and HER-2 mRNA expression for pathological complete response (pCR) in this setting. Interestingly, HER-2 gene expression was strongly predictive of pCR (P=0.017) as well as overall response (P=0.037) and clinical complete response (cCR, P=0.050). In contrast, for both TOPO IIalpha and MAPT no correlation with pCR was observed in our sample group.

[Identification of preeclampsia by cDNA-gene expression profiling in human placentas and serum -- a pilot study]. / Identifikation von Präeklampsiepatientinnen mittels cDNA-Genexpressionsprofilen an humanen Plazentaproben und Serum -- eine Pilotstudie.

OBJECTIVE: Preeclampsia is associated with significant maternal and fetal morbidity and mortality. The etiology remains unclear. For the accurate diagnosis and the prevention of preeclampsia it seems to be important to find a diagnostic tool that identifies risk patients before symptoms occur. With a new approach, the cDNA-Array analysis, human placentas and blood from preeclamptic and healthy pregnant women were examined for differentially expressed genes to find typical genes expression profiles. MATERIAL AND METHODS: In this pilot study, cDNA array analysis with a 19 200 gene array of placenta and blood samples from three preeclamptic patients have been performed to classify this samples based on expression patterns. RESULTS: Comparing normal placenta and blood from healthy delivered women (n = 4), a subset of 200 genes repeatedly found to be differentially expressed in preeclampsia. The placenta and blood samples from preeclampsia were accurately grouped by their individual gene expression patterns. CONCLUSIONS: These results suggest that the use of cDNA array is a tool to identify gene expression patterns in preeclampsia. With this set of differentially expressed genes in conjunction with sample clustering algorithms the identification of preeclampsia in placenta or blood samples is possible.

Gene expression profiles of breast cancer obtained from core cut biopsies before neoadjuvant docetaxel, adriamycin, and cyclophoshamide chemotherapy correlate with routine prognostic markers and could be used to identify predictive signatures.

BACKGROUND: Neoadjuvant administration of chemotherapy provides a unique opportunity to monitor response to treatment in breast cancer and assesses response exactly. Global gene expression profiling by microarrays has been used as a valuable tool for the identification of prognostic and predictive marker genes. Even though this technology is now wide spread and relatively standardized, there are only few data available which compare established parameters with expression values to determine reliability of this method. Therefore we analyzed gene expression data of pretreatment biopsies of breast cancer patients and compared them with the results of the immunohistochemical receptor expression for ER/ PR and Her-2, as well as FISH testing for HER-2 amplification. We analyzed the change of expression of these markers before and after neoadjuvant chemotherapy. Furthermore we evaluated the predictive significance of prognostic gene signatures as described by Sorlie, van't Veer and Ahr for response to neoadjuvant chemotherapy. METHODS: Pretherapeutic core biopsies were obtained from 70 patients undergoing neoadjuvant TAC chemotherapy within the GEPARTRIO-trial. Samples were characterized according to standard pathology including ER, PR and HER2 IHC and amount of cancer cells. Only biopsies with more than 80 % tumor cells were considered for further examination. RNA was isolated and expression profiling performed using Affymetrix Hg U133 Arrays (22 500 genes). GeneData's Expressionist software was used for bioinformatic analyses. RESULTS: More than two thirds of the biopsies yielded sufficient amounts (> 5 microg) of RNA for expression profiling and high quality data were obtained for 50 samples. Unsupervised clustering broadly revealed a correlation with hormone receptor status. When ER-alpha, PR and HER2 as analyzed by immunohistochemistry were compared to the corresponding mRNA data from gene chips more than 90 % concordance was observed. We could observe a switch of receptor expression for ER, PR or HER-2 from positive to negative and vice versa in 16/35 cases (45.7 %) and 5/22 cases (22.7 %) respectively. The prognostic marker sets of Sorlie, van't Veer and Ahr could not discriminate responders from non-responders in our patient group. CONCLUSIONS: Our results demonstrate that reliable expression profiles can be achieved by using limited amounts of tissue obtained during neoadjuvant chemotherapy. Microarray data capture conventional prognostic markers but might contain additional informative gene sets correlated with treatment outcome. Prognostic marker sets are not suitable to predict tumor response in the neoadjuvant setting, suggesting the necessity of class prediction methods to identify marker sets predictive for the type of therapy used.

Methylation of estrogen receptor beta promoter correlates with loss of ER-beta expression in mammary carcinoma and is an early indication marker in premalignant lesions.

The function of estrogen receptor beta (ER-beta) in mammary tissue is not completely understood. While early observations were often conflicting, more recent data suggest an important role as a tumor-suppressor gene. A decrease of ER-beta expression has been observed in ductal carcinoma in situ and invasive carcinoma as compared with benign mammary epithelial cells. The loss of ER-beta resulted in abnormal growth of mammary epithelial cells. We have previously shown that the mRNA expression of the ER-beta gene is almost totally suppressed in breast carcinomas from patients with a poor prognosis. Here we analyzed whether methylation changes in the different promoters of ER-beta are responsible for the loss of expression of the gene. A methylation assay with high specificity and sensitivity was developed, and a panel of breast tissue samples (n = 175) was characterized for methylation status. In contrast to benign breast, more than two-thirds of invasive breast cancers showed a high degree of methylation. Importantly, increased methylation was also detectable in numerous premalignant lesions. By analysis of breast tumors, previously characterized by gene-expression profiling, methylation was predominantly detected in a subgroup of patients with an unfavorable prognosis, suggesting a possible prognostic value of the ER-beta methylation status. We also investigated the structural characteristics of the two ER-beta promoters, which were both found to be closely associated with a second, downstream, localized and opposite-oriented promoter. However, we could not detect endogenous antisense RNA transcribed from these promoters, which may be involved in epigenetic gene silencing. We also failed to induce ER-beta promoter methylation by expressing siRNAs in cell lines. Interestingly, by comparing the promoter sequences of ER-beta with other genes known to be epigenetically inactivated in breast cancers, we identified a sequence motif possibly involved in promoter methylation.

Molecular classification of breast cancer patients by gene expression profiling.

For many tumors, pathological subclasses exist which have to be further defined by genetic markers to improve therapy and follow-up strategies. In this study, cDNA array analyses of breast cancers have been performed to classify tumors into categories based on expression patterns. Comparing purified normal ductal epithelial cells and corresponding tumour tissues, the expression of only a small fraction of genes was found to be significantly changed. A subset of genes repeatedly found to be differentially expressed in breast cancers was subsequently employed to perform a classification of 82 normal and malignant breast specimens by cluster analysis. This analysis identifies a subgroup of transcriptionally related tumours, designated class A, which can be further subdivided into A1 and A2. Correlation with classical clinicopathological parameters revealed that subgroup A1 was characterized by a high number of node-positive tumours (14 of 16). In this subgroup there was a disproportionate number of patients who had already developed distant metastases at the time of diagnosis (25% in this subgroup, compared with 5% among the rest of the samples). Taken together, the use of these differentially expressed marker genes in conjunction with sample clustering algorithms provides a novel molecular classification of breast cancer specimens, which facilitates the identification of patients with a higher risk of recurrence.

Adhesion induced expression of the serine/threonine kinase Fnk in human macrophages.

Members of the polo subfamily of protein kinases play crucial roles in cell proliferation. To study the function of this family in more detail, we isolated the cDNA of human Fnk (FGF-inducible kinase) which codes for a serine/threonine kinase of 646 aa. Despite the homology to the proliferation-associated polo-like kinase (Plk), tissue distribution of Fnk transcripts and expression kinetics differed clearly. In contrast to Plk no correlation between cell proliferation and Fnk gene expression was found. Instead high levels of Fnk mRNA were detectable in blood cells undergoing adhesion. The transition of monocytes from peripheral blood to matrix bound macrophages was accompanied by increasing levels of Fnk with time in culture. Neither treatment of monocytes with inducers of differentiation nor withdrawal of serum did influence Fnk mRNA levels significantly, suggesting that cell attachment triggers the onset of Fnk gene transcription. The idea that Fnk is part of the signalling network controlling cellular adhesion was supported by the analysis of the cytoplasmic distribution of the Fnk protein and the influence of its overexpression on the cellular architecture. Fnk as fusion protein with GFP localized at the cellular membrane in COS cells. Dysregulated Fnk gene expression disrupted the cellular f-actin network and induced a spherical morphology. Furthermore, Fnk binds to the Ca2+/integrin-binding protein Cib in two-hybrid-analyses and co-immunoprecipitation in assays. Moreover, both proteins were shown to co-localize in mammalian cells. The homology of Cib with calmodulin and with calcineurin B suggests that Cib might be a regulatory subunit of polo-like kinases.

PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.

Nef proteins of distinct HIV-1 or -2 isolates differ in their binding properties for HCK: isolation of a novel Nef binding factor with characteristics of an adaptor protein.

The Nef gene of the human and simian immunodeficiency viruses HIV and SIV has been implicated in pathogenicity; however, the mechanism by which Nef induces disease is still unknown. An impact on signal transduction in cells has been suggested by the interaction of Nef from an HIV-1 strain and tyrosine kinases like HCK and LCK as well as serine/threonine kinases. We have confirmed the binding of HCK to HIV-1 subtype B Nef and demonstrated an equally strong interaction with a subtype E Nef protein but weaker binding to Nef of HIV-2 subtype A (HIV-2D194). No binding, however, was observed to HIV-2 subtype B Nef (HIV-2D205). Instead, this protein bound to a novel cellular protein, Nefin 1, with characteristics of an adaptor protein and strong expression in all human hematopoietic tissues. Nefin 1 binds through an amino-terminal domain, which is related to SH3 domains. For interaction of Nef with Nefin 1, the PxxP motif and the three-dimensional conformation of the molecule appear necessary. In conclusion, this study demonstrates that Nef proteins of divergent strains of HIV-1 and HIV-2 may use different elements of signal transduction pathways for the induction of pathogenicity in vivo.

Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer.

Our previous data indicate that the expression of the PLK gene which codes for a serine/threonine kinase is restricted to proliferating cells. In Northern blot experiments PLK mRNA expression was at the limit of detection in normal lung tissue but elevated in most samples of non-small cell lung cancer (NSCLC). A very low frequency of PLK transcripts was only found in bronchiolo-alveolar carcinomas. NSCLC patients whose tumors showed moderate PLK expression survived significantly longer (5 year survival rate=51.8%) than those with high levels of PLK transcripts (24.2%, P=0.001). No statistically significant correlation was found between PLK mRNA expression and age, sex, TNM status, histological type or degree of differentiation. Interestingly, the prognosis of patients in post-surgical stages I and II was correlated with PLK expression (5 year survival rates in stage I: 69.1% (moderate PLK) - 43.5% (high PLK), P=0.03 or in stage II: 51.9% (moderate PLK) - 9.9% (high PLK), P=0.006). These results suggest that PLK mRNA expression provides a new independent prognostic indicator for patients with NSCLC.

Human SAK related to the PLK/polo family of cell cycle kinases shows high mRNA expression in testis.

We identified the nucleotide sequence of a cDNA encoding a polypeptide with a kinase domain that is related to the catalytic region of Drosophila melanogaster polo, Saccharomyces cerevisiae CDC5 as well as human FNK and PLK. The novel gene seems to represent the human counterpart of the mouse gene sak. The sequence of SAK predicts a serine/threonine kinase of 970 aa. The distribution of SAK mRNA in adult organs is restricted to certain tissues such as testis and thymus. Northern analyses of tumor tissues (lung, breast, brain) and corresponding normal tissues from the same patient did not reveal SAK expression. Comparing the mRNA distribution of the proliferation-associated polo-like kinase (PLK) with the expression of SAK we observed distinct differences. Thus, we suggest that these kinases have unique physiological roles in different cells or in response to different signals.