CD74 regulates complexity of tumor cell HLA class II peptidome in brain metastasis and is a positive prognostic marker for patient survival.

Despite multidisciplinary local and systemic therapeutic approaches, the prognosis for most patients with brain metastases is still dismal. The role of adaptive and innate anti-tumor response including the Human Leukocyte Antigen (HLA) machinery of antigen presentation is still unclear. We present data on the HLA class II-chaperone molecule CD74 in brain metastases and its impact on the HLA peptidome complexity.We analyzed CD74 and HLA class II expression on tumor cells in a subset of 236 human brain metastases, primary tumors and peripheral metastases of different entities in association with clinical data including overall survival. Additionally, we assessed whole DNA methylome profiles including CD74 promoter methylation and differential methylation in 21 brain metastases. We analyzed the effects of a siRNA mediated CD74 knockdown on HLA-expression and HLA peptidome composition in a brain metastatic melanoma cell line.We observed that CD74 expression on tumor cells is a strong positive prognostic marker in brain metastasis patients and positively associated with tumor-infiltrating T-lymphocytes (TILs). Whole DNA methylome analysis suggested that CD74 tumor cell expression might be regulated epigenetically via CD74 promoter methylation. CD74high and TILhigh tumors displayed a differential DNA methylation pattern with highest enrichment scores for antigen processing and presentation. Furthermore, CD74 knockdown in vitro lead to a reduction of HLA class II peptidome complexity, while HLA class I peptidome remained unaffected.In summary, our results demonstrate that a functional HLA class II processing machinery in brain metastatic tumor cells, reflected by a high expression of CD74 and a complex tumor cell HLA peptidome, seems to be crucial for better patient prognosis.

Urinary TIMP-1 and MMP-2 levels detect the presence of pancreatic malignancies.

BACKGROUND: A majority of patients with pancreatic malignancies, including both pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumours (pNETs), present with advanced disease due to a lack of specific symptoms and current diagnostic limitations, making this disease extremely difficult to detect. Our goal was to determine whether urinary matrix metalloproteases (uMMPs) and/or their endogenous inhibitors, urinary tissue inhibitor of metalloproteases (uTIMPs), could be detected in the urine of patients with pancreatic malignancies and whether they may serve as independent predictors of disease status. METHODS: Retrospective analyses of urine samples (n=139) from PDAC and pNET patients as well as age- and sex-matched controls were conducted. Urinary MMP-2 and uTIMP-1 levels were determined using ELISA and zymography. Biomarker expression in tumour and normal pancreatic tissues was analysed via immunohistochemistry (IHC). RESULTS: Multivariable logistic regression analyses indicated that, when controlling for age and sex, uMMP-2 (P<0.0001) and uTIMP-1 (P<0.0001) but not uMMP-9, were significant independent predictors for distinguishing between PDAC patients and healthy controls. Our data also indicated that uMMP-2 was an independent predictor of the presence of pNET. In addition, uTIMP-1 levels could differentiate the two cancer groups, PDAC and pNET, respectively. Immunohistochemistry analysis confirmed that MMP-2 and TIMP-1 protein expression is significantly upregulated in PDAC tissue compared with the normal pancreas. CONCLUSIONS: Taken together, our results suggest that the detection of uMMP-2 and uTIMP-1 may have diagnostic value in the detection of pancreatic malignancies and that uTIMP-1 may be useful in distinguishing between pancreatic adenocarcinoma and neuroendocrine tumours.

Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1).

The antibody trastuzumab (Herceptin) has substantially improved overall survival for patients with aggressive HER2-positive breast cancer. However, about 70% of all treated patients will experience relapse or disease progression. This may be related to an insufficient targeting of the CD44(high)CD24(low) breast cancer stem cell subset, which is not only highly resistant to chemotherapy and radiotherapy but also a poor target for trastuzumab due to low HER2 surface expression. Hence, we explored whether the new antibody-drug conjugate T-DM1, which consists of the potent chemotherapeutic DM1 coupled to trastuzumab, could improve the targeting of these tumor-initiating or metastasis-initiating cells. To this aim, primary HER2-overexpressing tumor cells as well as HER2-positive and HER2-negative breast cancer cell lines were treated with T-DM1, and effects on survival, colony formation, gene and protein expression as well as antibody internalization were assessed. This revealed that CD44(high)CD24(low)HER2(low) stem cell-like breast cancer cells show high endocytic activity and are thus particularly sensitive towards the antibody-drug conjugate T-DM1. Consequently, preexisting CD44(high)CD24(low) cancer stem cells were depleted by concentrations of T-DM1 that did not affect the bulk of the tumor cells. Likewise, colony formation was efficiently suppressed. Moreover, when tumor cells were cocultured with natural killer cells, antibody-dependent cell-mediated cytotoxicity was enhanced, and EMT-mediated induction of stem cell-like properties was prevented in differentiated tumor cells. Thus our study reveals an unanticipated targeting of stem cell-like breast cancer cells by T-DM1 that may contribute to the clinical efficacy of this recently approved antibody-drug conjugate.

Downregulation of AKT reverses platinum resistance of human ovarian cancers in vitro.

Platinum resistance is the most crucial problem for treatment of ovarian cancer. Increasing evidence points towards AKT overexpression as a mechanistic reason for this clinical condition. The present study evaluates the effect of overexpression and downregulation of AKT on the sensitivity to cisplatin in a platinum-resistant human ovarian cancer cell line and the corresponding platinum-sensitive parental cell line. A2780 and A2780cis ovarian cancer cell lines were stably transfected with an AKT-sense and AKT-antisense plasmid. Successful transfection was evaluated by western blot analysis. Cytotoxic effects of cisplatin were evaluated by metabolic (MTT) and clonogenicity assays as well as by FACS analysis. AKT overexpression (confirmed by western blotting) converted platinum-sensitive A2780 into platinum-resistant cells as shown by MTT assay. Importantly, platinum resistance of A2780cis cells could be reversed by downregulation of AKT, as demonstrated by MTT and clonogenicity assays and FACS analysis. Our data provide strong evidence that cisplatin resistance in ovarian cancer is mediated by AKT overexpression and can be overcome by AKT downregulation, thus, providing a rationale for clinical phase II/III studies combining AKT inhibitors with cisplatin.

The post-reproductive Fallopian tube: better removed?

Recently, the distal Fallopian tube has attracted considerable attention not only as site of origin for serous cancer in women with BRCA mutations, but also as the anatomical location where the majority of serous ovarian cancers apparently develop. Consequently, the Fallopian tube may be the unique location where early 'ovarian' cancers can be found--which would contradict the long-standing impression that the ovaries and the Fallopian tubes are always simultaneously involved. Based on the dismal prognosis associated with ovarian cancer and our inability to screen for early-stage disease, we discuss the rationale for introducing salpinges-hysterectomy as new clinical standard for women in need of hysterectomy and further weigh the arguments for and against bilateral salpingectomy as a sterilization method. There is no known physiological benefit of retaining the post-reproductive Fallopian tube during hysterectomy or sterilization, especially as this does not affect ovarian hormone production. On the other hand, the consequences associated with a surgical menopause provide a rationale for preserving the ovaries in premenopausal women. Prophylactic removal of the Fallopian tubes during hysterectomy or sterilization would rule out any subsequent tubal pathology, such as hydrosalpinx, which is observed in up to 30% of women after hysterectomy. Moreover, this intervention is likely to offer considerable protection against later tumour development, even if the ovaries are retained. Thus, we recommend that any hysterectomy should be combined with salpingectomy. In addition, women over 35 years of age could also be offered bilateral salpingectomy as means of sterilization.

Whole blood-derived miRNA profiles as potential new tools for ovarian cancer screening.

BACKGROUND: Screening is an unsolved problem for ovarian cancer (OvCA). As late detection is equivalent to poor prognosis, we analysed whether OvCA patients show diagnostically meaningful microRNA (miRNA) patterns in blood cells. METHODS: Blood-borne whole miRNome profiles from 24 patients with OvCA and 15 age- and sex-matched healthy controls were biostatistically evaluated. RESULTS: Student's t-test revealed 147 significantly deregulated miRNAs before and 4 after Benjamini-Hochberg adjustment. Although these included miRNAs already linked to OvCA (e.g., miR-16, miR-155), others had never before been connected to specific diseases. A bioinformatically calculated miRNA profile allowed for discrimination between blood samples of OvCA patients and healthy controls with an accuracy of >76%. When only cancers of the serous subtype were considered and compared with an extended control group (n=39), accuracy, specificity and sensitivity all increased to >85%. CONCLUSION: Our proof-of-principle study strengthens the hypothesis that neoplastic diseases generate characteristic miRNA fingerprints in blood cells. Still, the obtained OvCA-associated miRNA pattern is not yet sensitive and specific enough to permit the monitoring of disease progression or even preventive screening. Microarray-based miRNA profiling from peripheral blood could thus be combined with other markers to improve the notoriously difficult but important screening for OvCA.

Stem cells, melanoma and cancer stem cells: the good, the bad and the evil?

Most cancers contain morphologically heterogeneous populations of cells. While this observation may partly be explained by the coexistence of multiple genetic sub-clones arising through independent somatic mutations and/or as a result of differentiation processes in the tumor microenvironment, it also implies that the tumor may be formed from undifferentiated ''stem cell-like'' cells called ''cancer stem cells'' or ''cancer-initiating cells''. These cells are thought to constitute one or several rare subpopulations in a given tumor and would be strongly responsible for initiation of tumor development and growth as well as for metastasis and recurrence after cytoreductive therapy. However, while the concept of cancer stem cells has been first established for human myeloid leukemia in the 1960s, it has only much later been extended to other solid tumors such as breast or brain cancers and most recently to melanoma. Thus, it is presently unclear which role a sufficiently characterized population of melanoma stem cells plays in cancer promotion and progression. Here, we review the emerging melanoma stem cell model and discuss the biological and therapeutic implications of the model.

A novel p53 rescue compound induces p53-dependent growth arrest and sensitises glioma cells to Apo2L/TRAIL-induced apoptosis.

Reactivation of mutant p53 in tumours is a promising strategy for cancer therapy. Here we characterise the novel p53 rescue compound P53R3 that restores sequence-specific DNA binding of the endogenously expressed p53(R175H) and p53(R273H) mutants in gel-shift assays. Overexpression of the paradigmatic p53 mutants p53(R175H), p53(R248W) and p53(R273H) in the p53 null glioma cell line LN-308 reveals that P53R3 induces p53-dependent antiproliferative effects with much higher specificity and over a wider range of concentrations than the previously described p53 rescue drug p53 reactivation and induction of massive apoptosis (PRIMA-1). Furthermore, P53R3 enhances recruitment of endogenous p53 to several target promoters in glioma cells bearing mutant (T98G) and wild-type (LNT-229) p53 and induces mRNA expression of numerous p53 target genes in a p53-dependent manner. Interestingly, P53R3 strongly enhances the mRNA, total protein and cell surface expression of the death receptor death receptor 5 (DR5) whereas CD95 and TNF receptor 1 levels are unaffected. Accordingly, P53R3 does not sensitise for CD95 ligand- or tumour necrosis factor alpha-induced cell death, but displays synergy with Apo2L.0 in 9 of 12 glioma cell lines. Both DR5 surface induction and synergy with Apo2L.0 are sensitive to siRNA-mediated downregulation of p53. Thus this new p53 rescue compound may open up novel perspectives for the treatment of cancers currently considered resistant to the therapeutic induction of apoptosis.

The role of regulatory T cells in ovarian cancer.

Regulatory T cells (T(reg)), also termed suppressor T cells, control self-reactive T cells in the periphery, thereby conferring protection against immunologic self-destruction. While T(reg) are essential for the prevention of autoimmunity, they also inhibit immune responses against tumor antigens. This is corroborated by an increased mortality rate associated with the presence of a high number of intratumoral T(reg). Tumor infiltration by non-T(reg), on the other hand, is predictive for a substantially longer patient survival. These clinical data suggest that ovarian cancer patients can spontaneously mount effective antitumor immune responses that are undermined by T(reg)-mediated tolerization. The present article reviews clinical and experimental findings on T(reg) in ovarian cancer, with special regard to potential therapeutic implications, which may result from the existing evidence.

PCTAIRE3: a putative mediator of growth arrest and death induced by CTS-1, a dominant-positive p53-derived synthetic tumor suppressor, in human malignant glioma cells.

Chimeric tumor suppressor-1 (CTS-1) is based on the sequence of p53 and was designed as a therapeutic tool resisting various mechanisms of p53 inactivation. We previously reported that an adenovirus expressing CTS-1 (Ad-CTS-1) has superior cell death-inducing activity in glioma cells compared with wild-type p53. Here, we used cDNA microarrays to detect changes in gene expression preferentially induced by Ad-CTS-1. The putative serine threonine kinase, PCTAIRE3, and the quinone oxireductase, PIG3, were strongly induced by Ad-CTS-1 compared with wild-type p53. An adenoviral vector encoding PCTAIRE3 (Ad-PCTAIRE3) induced growth arrest and killed a minor proportion of the glioma cells. Ad-PIG3 alone affected neither growth nor viability. However, coinfection with Ad-PCTAIRE3 and Ad-PIG3 resulted in enhanced growth inhibition compared with Ad-PCTAIRE3 infection alone. Ad-CTS1, Ad-PCTAIRE3 or Ad-PIG3 induced the formation of free reactive oxygen species (ROS). However, the prevention of ROS formation induced by Ad-PCTAIRE3 and Ad-CTS-1 did not block growth arrest and cell death, suggesting that ROS formation is not essential for these effects. Altogether, these data identify PCTAIRE3 as one novel growth-inhibitory and death-inducing p53 response gene and suggest that changes in the expression of specific target genes contribute to the superior anti-glioma activity of CTS-1.

Alkylphosphocholine-induced glioma cell death is BCL-X(L)-sensitive, caspase-independent and characterized by massive cytoplasmic vacuole formation.

Alkylphosphocholines (APC) are candidate anticancer agents. We here report that APC induce the formation of large vacuoles and typical features of apoptosis in human glioma cell lines, but not in immortalized astrocytes. APC promote caspase activation, poly(ADP-ribose)-polymerase (PARP) processing and cytochrome c release from mitochondria. Adenoviral X-linked inhibitor of apoptosis (XIAP) gene transfer, or exposure to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoro-methylketone zVAD-fmk, blocks caspase-7 and PARP processing, but not cell death, whereas BCL-X(L) blocks not only caspase-7 and PARP processing but also cell death. APC induce changes in Delta Psi m in sensitive glioma cells, but not in resistant astrocytes. The changes in Delta Psi m are unaffected by crm-A (cowpox serpin-cytokine response modifier protein A), XIAP or zVAD-fmk, but blocked by BCL-X(L), and are thus a strong predictor of cell death in response to APC. Free radicals are induced, but not responsible for cell death. APC thus induce a characteristic morphological, BCL-X(L)-sensitive, apparently caspase-independent cell death involving mitochondrial alterations selectively in neoplastic astrocytic cells.

CP-31398, a novel p53-stabilizing agent, induces p53-dependent and p53-independent glioma cell death.

CP-31398 is a prototype small molecule that stabilizes the active conformation of p53 and promotes p53 activity in cancer cell lines with mutant or wild-type p53. Here, we report that CP-31398 induces p53 reporter gene activity and p21 expression in all of 11 glioma cell lines harboring wild-type or mutant p53, but not in p53-null LN-308 cells. Upon prolonged exposure to CP-31398, all glioma cell lines undergo caspase-independent and bcl-x(L)-insensitive cell death with EC(50) concentrations of 10-36 microM. By comparing p53 wild-type U87MG and p53-null LN-308 cells expressing the temperature-sensitive p53(V135A) mutant, we delineate two pathways of CP-31398-induced cell death: an early, p53-dependent pathway that requires (new p53) protein synthesis and a late, p53-independent pathway characterized by aurintricarboxylic acid -sensitive calcium release and epiphenomenal free radical formation. Post-transcriptional repression of p53 synthesis by an intracellularly transcribed short interfering RNA confirmed the presence of these two pathways of cell death. These observations point out some of the liabilities of CP-31398 as a prototype p53-based therapeutic and define a rationale for further refinement of small molecules that specifically target the p53 pathway, but lack the p53-independent effects.

N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) suppresses microglial inducible nitric oxide synthase (iNOS) expression and activity induced by interferon-gamma (IFN-gamma).

1. Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-inflammatory stimuli including interferon-gamma (IFN-gamma), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive effects on the activation of monocytes. 2. Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-gamma (200 u x ml(-1)) as measured by Northern and Western blot analyses and Griess assay. 3. Exposure to non-toxic doses of tranilast (30-300 microM) leads to a concentration-dependent inhibition of IFN-gamma-induced (200 u x ml(-1)) iNOS mRNA and protein expression. This is paralleled by a suppression of NO-release into the cell culture medium. 4. Inhibition of IFN-gamma-induced iNOS mRNA expression by tranilast is paralleled by an inhibition of nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of inhibitory kappaB (IkappaB) as determined by Western blot analyses and NF-kappaB reporter gene assay. 5. These results suggest that tranilast-mediated suppression of microglial iNOS activity induced by IFN-gamma involves the inhibition of NF-kappaB-dependent iNOS mRNA expression.

Diva/Boo is a negative regulator of cell death in human glioma cells.

Diva is a novel proapoptotic member of the Bcl-2 protein family which binds apoptosis activating factor-1 (APAF-1). Diva is identical with Boo which was identified as a novel antiapoptotic Bcl-2 family protein. Here, we report that Diva promotes the cell cycle exit of human glioma cells in response to serum deprivation and inhibits apoptosis of these cells induced by CD95 ligand or chemotherapeutic drugs. In glioma cells, Diva interferes with apoptotic signaling downstream of cytochrome c release, but upstream of caspase activation, consistent with an inhibitory effect on the mitochondrial amplification step involving the apoptosome and APAF-1.

Alcohol enhances oxysterol-induced apoptosis in human endothelial cells by a calcium-dependent mechanism.

Controversy exists about the net effect of alcohol on atherogenesis. A protective effect is assumed, especially from the tannins and phenolic compounds in red wine, owing to their inhibition of low density lipoprotein (LDL) oxidation. However, increased atherogenesis occurs in subjects with moderate to heavy drinking habits. The purpose of this study was to investigate the influence of alcohol in combination with oxysterols on the endothelium. Cultured human arterial endothelial cells (HAECs) served as an in vitro model to test the cellular effects of various oxysterols. Oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and cholesterol-5,6-epoxides), which are assumed to be the most toxic constituents of oxidized LDL, induced apoptosis in HAECs through calcium mobilization followed by activation of caspase-3. Ethanol, methanol, isopropanol, tert-butanol, and red wine all potentiated oxysterol-induced cell death up to 5-fold, paralleled by further induction of caspase-3. The alcohol effect occurred in a dose-dependent manner and reached a plateau at 0.05% concentration. Alcohol itself did not affect endothelial cell viability, nor did other solvents such as dimethyl sulfoxide mimic the alcohol effect. So far as the physiologically occurring oxysterols are concerned, this effect was apparent only for oxysterols oxidized at the steran ring. The possibility of alcohol facilitating the uptake of oxysterols into the cell was not supported by the data from an uptake study with radiolabeled compounds. Finally, alcohol in combination with oxysterols did cause a dramatic increase in cytosolic calcium influx. Blockage of calcium influx by the calcium channel blocker aurintricarboxylic acid or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abrogated the alcohol-mediated enhancement of oxysterol toxicity. We describe for the first time a mechanistic concept explaining possible adverse effects of alcohol in conjunction with physiologically occurring oxysterols on atherogenesis.

Glioma cell sensitivity to topotecan: the role of p53 and topotecan-induced DNA damage.

Topotecan is a topoisomerase I inhibitor which is currently evaluated as an adjuvant agent for malignant glioma. Here, we analysed the effects of topotecan on 12 human malignant glioma cell lines in vitro. All cell lines expressed topoisomerase I mRNA. High p53 protein levels, but not genetic or functional p53 status, were associated with increased topotecan-induced DNA/topoisomerase I complex formation. Neither functional p53 status, nor p53 protein levels, nor complex formation predicted topotecan-induced growth inhibition. We thus confirm a possible role for p53 protein in modulating topoisomerase I activity but conclude that the major molecular determinants of topotecan sensitivity in glioma cells await identification.

Identification by suppression subtractive hybridization of p21 as a radio-inducible gene in human glioma cells.

BACKGROUND: Radio-gene therapy involves the delivery, to tumor cells, of a therapeutic transgene whose expression is controlled by irradiation. MATERIALS AND METHODS: Here, we sought to identify novel radio-inducible transcripts in U87MG human malignant glioma cells using suppression subtractive hybridization (SSH). RESULTS: Of 998 clones from a subtracted library of irradiated U87MG cells, 24 candidate clones were identified by dot blot and 3 clones were confirmed as having been induced by irradiation by Northern blot analysis. All three clones showed 99-100% homology to the cyclin-dependent kinase (cdk) inhibitor, p21(Waf/Cip1). A screening of 12 human malignant glioma cell lines revealed that irradiation increased p21 mRNA expression and p21 protein levels levels in all of the five cell lines retaining p53 wild-type activity in a p53 reporter assay, but in none of seven p53 reporter-negative cell lines. CONCLUSION: Irradiation induces p21 mRNA expression in a strictly p53-dependent manner and may only enhance the expression of a limited number of genes in glioma cells. We conclude that the identification of radio-inducible genomic sequences suitable for radio-gene therapy may turn out to be difficult.