MicroRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495 and miR-654-3p, mapped to the 14q32.31 locus, regulate proliferation, apoptosis, migration and invasion in metastatic prostate cancer cells.

miRNAs act as oncogenes or tumor suppressors in a wide variety of human cancers, including prostate cancer (PCa). We found a severe and consistent downregulation of miRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495 and miR-654-3p, mapped to the 14q32.31 region in metastatic cell lines as compared with normal prostatic epithelial cells (PrEC). In specimens of human prostate (28 normals, 99 primary tumors and 13 metastases), lower miRNA levels correlated significantly with a higher incidence of metastatic events and higher prostate specific antigen (PSA) levels, with similar trends observed for lymph node invasion and the Gleason score. We transiently transfected 10 members of the 14q32.31 cluster in normal prostatic epithelial cell lines and characterized their affect on malignant cell behaviors, including proliferation, apoptosis, migration and invasion. Finally, we identified FZD4, a gene important for epithelial-to-mesenchymal transition in (PCa), as a target of miR-377.

miR-143 regulates hexokinase 2 expression in cancer cells.

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3'-untranslated region (3'UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3'UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

DNA methylation silences miR-132 in prostate cancer.

Silencing of microRNAs (miRNAs) by promoter CpG island methylation may be an important mechanism in prostate carcinogenesis. To screen for epigenetically silenced miRNAs in prostate cancer (PCa), we treated prostate normal epithelial and carcinoma cells with 5-aza-2'-deoxycytidine (AZA) and subsequently examined expression changes of 650 miRNAs by megaplex stemloop reverse transcription-quantitative PCR. After applying a selection strategy, we analyzed the methylation status of CpG islands upstream to a subset of miRNAs by methylation-specific PCR. The CpG islands of miR-18b, miR-132, miR-34b/c, miR-148a, miR-450a and miR-542-3p showed methylation patterns congruent with their expression modulations in response to AZA. Methylation analysis of these CpG islands in a panel of 50 human prostate carcinoma specimens and 24 normal controls revealed miR-132 to be methylated in 42% of human cancer cases in a manner positively correlated to total Gleason score and tumor stage. Expression analysis of miR-132 in our tissue panel confirmed its downregulation in methylated tumors. Re-expression of miR-132 in PC3 cells induced cell detachment followed by cell death (anoikis). Two pro-survival proteins-heparin-binding epidermal growth factor and TALIN2-were confirmed as direct targets of miR-132. The results of this study point to miR-132 as a methylation-silenced miRNA with an antimetastatic role in PCa controlling cellular adhesion.

Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer.

BACKGROUND: MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker. METHODS: The 5-aza-2'-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a. RESULTS: The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561. CONCLUSIONS: These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker.

Immunotoxin-mediated inhibition of chronic lymphocytic leukemia cell proliferation in humans.

We evaluated the cytotoxicity of the immunotoxin OKT1-SAP on fresh B-chronic lymphocytic leukemia (B-CLL) cells from 31 consecutive patients. OKT1-SAP comprised the OKT1 (CD5) monoclonal antibody disulfide linked to saporin-6 (SAP) ribosome-inactivating protein from the plant Saponaria officinalis. The effect of OKT1-SAP on target CD5-positive B-CLL cells was estimated using an in vitro proliferation inhibition assay in which control or OKT1-SAP-treated B-CLL cells were induced to proliferate by sequential stimulation with insolubilized anti-C3b receptor CB04 (CD35) antibody and low molecular weight B-cell growth factor. In 90% of patients, OKT1-SAP specifically suppressed B-CLL cell proliferation in a dose-related manner (50% inhibitory concentration, 4.0-6.8 nM). Taken together the findings reported in this article provide information relevant to the clinical development of immunotoxins because: (a) the in vitro conditions under which B-CLL cell proliferation is inhibited by OKT1-SAP are achievable in vivo without nonspecific toxicity according to our previous toxicology and pharmacokinetics studies in primates; and (b) the B-CLL cell proliferation inhibition assay described here provides a basis for future comparative studies.

In vivo effects in mice of an anti-T cell immunotoxin.

We have evaluated both the proliferative response as well as the Thy-1 Ag expression of lymphocytes from mice treated in vivo with an anti-Thy-1 immunotoxin (IT). The IT was a rat IgG2c mAb recognizing the Thy-1 Ag, disulfide-linked to a ribosome-inactivating protein isolated from the seeds of the plant Saponaria officinalis (soapwort). Toxicity studies showed that a single i.v. injection of doses up to 20 micrograms IT/mouse was well tolerated and allowed indefinite survival. The Con A-induced proliferative response of spleen cells from mice killed 1 day after treatment with sublethal doses of IT was inhibited in a dose-dependent manner, with complete inhibition observed at doses of greater than or equal to 5 micrograms IT/mouse. Control experiments showed that the inhibition was due to the IT and not to its single components. Moreover, the IT effect was abolished by a large (100-fold) excess of anti-Thy-1 mAb alone given concurrently, but not by an unrelated, isotype-matched rat mAb. At all IT doses, the proliferative response to a B cell mitogen (LPS) was normal. Kinetic studies showed a time- and dose-dependent reconstitution of Con A responsiveness. In limiting dilution cultures of spleen cells from mice treated with 5 micrograms IT 1 or 4 days before death, a 97% depletion of T lymphocytes capable of proliferation was observed. Limiting dilution cultures showed that also the thymus of IT-treated mice was depleted by more than 90% of growth-competent T lymphocytes. Cytofluorographic studies of Thy-1+ cells from the spleens of IT-treated mice gave results which did not correlate with those obtained in functional assays. Thus, a dose-dependent reduction, followed by a time-dependent reconstitution of Thy-1+ cells was observed in this case too, but the depletion occurred at later time points and was less complete than that observed in functional assays. Moreover, the mean fluorescence intensity of the residual Thy-1+ cells decreased below normal levels.

Immunotoxin mediated killing of clonable T-lymphocytes infiltrating an irreversibly rejected human renal allograft.

We isolated and treated in vitro with a novel CD5-specific saporin immunotoxin, referred to as OKT1-SAP, the cells infiltrating an irreversibly rejected renal allograft from a patient who rejected while on cyclosporine plus steroids and then failed to respond to multiple courses of high-dose steroids, intravenous OKT3 antibody, and local irradiation to the graft. We report here that under experimental conditions achievable in vivo the immunotoxin OKT1-SAP was capable of eliminating in vitro more than 95% of clonable T-lymphocytes infiltrating the rejected allograft of this patient despite their resistance to previous aggressive immunosuppression. To our knowledge, this is the first report of an immunotoxin-mediated suppression of the clonogenic growth of rejected renal allograft infiltrating T-lymphocytes.

B-cell restricted saporin immunotoxins: activity against B-cell lines and chronic lymphocytic leukemia cells.

B cell-restricted immunotoxins were constructed by conjugating anti-B monoclonal antibodies to saporin, the major ribosome inactivating protein from the seeds of the plant Saponaria officinalis. HD37-SAP is directed against CD19, the broadest B cell-specific determinant. HD39-SAP and HD6-SAP recognize two different epitopes on the CD22 molecule, an antigen present on the cell surface of B cells at late stages of differentiation. All three immunotoxins inhibited DNA synthesis and protein synthesis in target B lymphoma cells with a dose-related effect, in short incubation times and in the absence of potentiators. A clonogenic assay demonstrated that all immunotoxins could eliminate more than two logs of clonogenic malignant B cells with a two-hour incubation at concentrations not toxic to cells not bearing target antigens. The immunotoxin activity was evaluated by DNA synthesis inhibition in fresh B-chronic lymphocytic leukemia cells (B-CLL) stimulated to proliferate by incubation with an antibody specific for the receptor of C3b complement component (CR1) plus B cell growth factor. B-CLL cell DNA synthesis was actively inhibited by treatment at low immunotoxin concentration without need of potentiators. Immunotoxins exerted their effect also in whole blood of CLL patients under conditions achievable in vivo. We conclude that B cell-restricted immunotoxins HD37-SAP, HD39-SAP, and HD6-SAP are good candidates for in vivo therapy of B-cell malignancies.

Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation.

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.

Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates.

The authors conjugated, by a disulphide bond, the antihuman T-lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen-induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double-antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.

Activity of a monoclonal antibody-saporin-6 conjugate against B-lymphoma cells.

A monoclonal antibody reactive with the immunoglobulin heavy chain (TEC IgM) has been conjugated to saporin-6 (SAP), which is the major ribosome-inactivating protein from the seeds of the plant Saponaria officinalis. Studies with Burkitt's lymphoma cell line Bjab 113 demonstrate that this immunotoxin is capable of killing 3 logs (99.9%) of clonogenic lymphoma cells after a 2-hour incubation. The presence of human bone marrow inhibits the activity of the conjugate. However, full potency of TEC IgM-SAP immunotoxin is restored by adding 1 mM amantadine to the incubation medium. The reaction is highly specific and is inhibited by the presence of excess anti-mu-antibody or human serum. Clonal growth of other Burkitt's lymphoma cell lines is inhibited to a lesser extent by the immunotoxin. The presence of surface IgM on the different cell lines is directly correlated to target cell killing by TEC IgM-SAP. Isolation of Bjab 113 clones surviving treatment demonstrates that only a minority are truly resistant and that the others randomly escape the treatment. The highly potent and specific activity of this conjugate in the presence of bone marrow buffy coat and its exceptionally rapid onset of action make this conjugate a good candidate for the ex vivo elimination of neoplastic cells from the bone marrow of non-Hodgkin's lymphoma patients.

Immunological studies on the origin of the precursor cells in Waldenström's macroglobulinemia.

Preliminary results obtained with OK anti-T and FMC anti-B monoclonal antibodies in a study of peripheral blood lymphocytes from eight patients with Waldenström's macroglobulinemia and of bone marrow lymphocytes from three patients, are reported. Endocytoplasmic immunofluorescence revealed a 1 to 3% marrow plasma cell fraction in all three cases, together with an approximately 10% increase in the monoclonal precursor compartment. Displacement of the peripheral blood helper: suppressor ratio was also observed using anti-T-lymphocyte monoclonal antibodies.