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Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and rapidly lethal tumor, poorly responsive to conventional treatments. In this regards, the identification of molecular alterations underlying DMPM onset and progression might be exploited to develop novel therapeutic strategies. Here, we focused on miR-550a-3p, which we found downregulated in 45 DMPM clinical samples compared to normal tissues and whose expression levels were associated with patient outcome. Through a gain-of-function approach using miRNA mimics in 3 DMPM cell lines, we demonstrated the tumor-suppressive role of miR-550a-3p. Specifically, miRNA ectopic expression impaired cell proliferation and invasiveness, enhanced the apoptotic response, and reduced the growth of DMPM xenografts in mice. Antiproliferative and proapoptotic effects were also observed in prostate and ovarian cancer cell lines following miR-550a-3p ectopic expression. miR-550a-3p effects were mediated, at least in part, by the direct inhibition of HSP90AA1 and the consequent downregulation of its target proteins, the levels of which were rescued upon disruption of miRNA-HSP90AA1 mRNA pairing, partially abrogating miR-550a-3p-induced cellular effects. Our results show that miR-550a-3p reconstitution affects several tumor traits, thus suggesting this approach as a potential novel therapeutic strategy for DMPM.

Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts). E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (Kd imm) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and—in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer—cellular assays. Taken together, the success of our strategy—which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry—in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.

We evaluated the effects of the trinuclear platinum complex, BBR 3464, in a human ovarian carcinoma cell line (OAW42) and in its cisplatin (CDDP)-resistant counterpart (OAW42MER). A 14-fold increased sensitivity to a 1-h BBR 3464 exposure was found in OAW42MER cells compared with their parental cell line. Flow cytometric experiments showed that BBR 3464 was able to induce a persistent block of OAW42 and OAW42MER cells in the G 2 M phase, whereas CDDP caused an initial accumulation of cells in the S phase followed by an increase in the G 2 M cell fraction in both cell lines. Exposure to equitoxic (IC 50 ) drug concentrations induced programmed cell death in both cell lines. However, the percentage of cells with an apoptotic nuclear morphology was slightly higher after CDDP than BBR 3464 treatment in OAW42 cells, whereas the opposite pattern was observed in OAW42MER cells. Degradation of the nuclear lamin B was detected in OAW42 cells after exposure to each drug. Conversely, in OAW42MER cells lamin B cleavage was only appreciable after BBR 3464 exposure. In OAW42 cells, CDDP and BBR 3464 did not appreciably affect the mitochondrial membrane potential (Δψ mt ), whereas in the OAW42MER cell line a marked Δψ mt reduction was observed after exposure to BBR 3464, but not to CDDP. The results of the study would suggest that the sensitivity to BBR 3464 observed in the CDDP-resistant OAW42MER cell line might be attributable to the ability of the trinuclear platinum complex to modify DNA in a way which is different from that of CDDP and, as a consequence, to induce different cellular responses to DNA damage such as the triggering of specific apoptotic pathways.

Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. No telomere shortening, however, was observed in these clones. Overall, our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors.

By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders. This protein binds with very high affinity AT containing DNA treated with minor groove binders such as distamycin A, Hoechst 33258 and 33342, CC-1065 and ethidium bromide minor groove intercalator, but not with major groove binders such as quinacrine mustard, cisplatin or melphalan, or with topoisomerase I inhibitor camptothecin or topoisomerase II inhibitor doxorubicin. This protein was found to be present in different extracts of human, murine and hamster cells, with the human protein which appears to have a molecular weight slightly lower than that of the other species. This protein was found to be expressed both in cancer and normal tissues. By using molecular ultrafiltration techniques as well as southwestern analysis it was estimated that the apparent molecular weight is close to 100 kDa. We can exclude an identity between this protein and other proteins, with a similar molecular weight previously reported to be involved in DNA damage recognition/repair, such as topoisomerase I, mismatch repair activities such as the prokaryotic MutS protein and its human homologue hMSH2 or proteins of the nucleotide excision repair system such as ERCC1, -2, -3 and -4.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which has been implicated in inhibition of apoptosis and control of mitotic progression. The finding that survivin is overexpressed in most human tumors but absent in normal adult tissues has led to the proposal of survivin as a promising therapeutic target for anticancer therapies. We decided to evaluate the effects of a ribozyme-based strategy for survivin inhibition in androgen-independent human prostate cancer cells. We constructed a Moloney-based retroviral vector expressing a ribozyme targeting the 3' end of the CUA(110) triplet in survivin mRNA, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations obtained by infection with the retroviral vector of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were selected for the study. Ribozyme-expressing prostate cancer cells were characterized by a significant reduction of survivin expression compared to parental cells transduced with a control ribozyme; the cells became polyploid, underwent caspase-9-dependent apoptosis and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of prostate cancer cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted in athymic nude mice. These findings suggest that manipulation of the antiapoptotic survivin pathway may provide a novel approach for the treatment of androgen-independent prostate cancer.

Survivin is a structurally unique member of the inhibitors of apoptosis protein family and is involved in the control of cell division and inhibition of apoptosis. The notion that survivin is overexpressed in most human tumors but absent in normal adult tissues with only a few exceptions has led to the proposal of survivin as a promising therapeutic target for novel anticancer therapies. In this context, we generated a hammerhead ribozyme targeting the 3' end of the CUA110 triplet in the survivin mRNA. Two human melanoma cell lines (JR8 and M14) overexpressing survivin were stably transfected with the pRc/CMV vector carrying the ribozyme sequence. Two polyclonal cell populations proven to endogenously express ribozyme and characterized by a markedly lower survivin protein level (-60% and -50%, respectively) than JR8 and M14 parental cells were selected for the study. Ribozyme-expressing cells showed a significantly (p0.01) increased sensitivity to gamma-irradiation (as detected by clonogenic cell survival) compared to JR8 and M14 cells. Moreover, in the JR8 cell line, the extent of radiation-induced apoptosis (in terms of percentage of apoptotic nuclei in cells stained with propidium iodide and level of caspase-3 catalytic activity) was markedly greater in ribozyme-expressing cells than in parental cells. These results demonstrate for the first time that attenuation of survivin expression renders human melanoma cells more susceptible to gamma-irradiation.

Stable transfection of human ovarian carcinoma cells with survivin cDNA caused a four- to sixfold increase in cell resistance to taxotere and taxol (two-sided Student's t test, p0.05), with a concomitant reduction in the apoptotic response to taxol, but did not affect cell sensitivity to cisplatin or oxaliplatin. Such findings were indirectly supported by similar observations obtained with clinical tumours. In fact, high levels of survivin protein expression (30% positive cells), detected by immunohistochemistry in 90/124 (73%) advanced ovarian carcinomas, were significantly associated with clinical resistance to a taxol/platinum-based regimen but unrelated to tumour shrinkage following cisplatin-including combinations (non-taxol based). In the 95 patients receiving a taxol/platinum-based regimen, survivin overexpression correlated with a lower clinical or pathologic complete remission rate than absent/low protein expression (43 vs 75%, p = 0.0058 by logistic regression adjusted for tumour stage, histological grade and p53 expression). Conversely, in the 29 cases treated with cisplatin-containing regimens (not taxol based), survivin expression was unrelated to tumour response. Cellular studies and clinical data suggest a direct link between survivin expression and tumour cell susceptibility to taxol.

Some DNA minor groove binders have shown antiviral and antitumor activity. The distamycin derivative Tallimustine and the CC-1065 derivative Adozelesin and Carzelesin are under clinical investigationin phase II clinical trials. The mechanisms of repair of the DNA lesions caused by these drugs are unknown. By using the gel retardation assay, we have identified a protein able to recognize a fragment of DNA only if it was previously reacted with minor groove binders (MGB). This protein binds with very high affinity AT containing DNA treated with MGB such as Distamycin A or Hoechst 33258 but not with a major groove binders (such as e.g. quinacrine mustard). This protein was found to be present in extracts of human, murine or hamster cells, although the human protein appears to have a molecular weight slightly lower than that of the other species. This protein is expressed both in cancer and normal tissues. By using ultrafiltration techniques as well as Southwestern analysis it was estimated that the apparent molecular weight is close to 100 Kda. Work is in progress to purify this protein which might be involved in the recognition and repair of DNA damage caused by minor groove binders.