Impairment of the immune response and aberrant expression of microRNAs are emerging hallmarks of tumour initiation/progression, in addition to driver gene mutations and epigenetic modifications. We ...performed a preliminary survey of independent adenoma and colorectal cancer (CRC) miRnoma data sets and, among the most dysregulated miRNAs, we selected miR-27a and disclosed that it is already upregulated in adenoma and further increases during the evolution to adenocarcinoma. To identify novel genes and pathways regulated by this miRNA, we employed a differential 2DE-DIGE proteome analysis. We showed that miR-27a modulates a group of proteins involved in MHC class I cell surface exposure and, mechanistically, demonstrated that calreticulin is a miR-27a direct target responsible for most downstream effects in epistasis experiments. In vitro miR-27a affected cell proliferation and angiogenesis; mouse xenografts of human CRC cell lines expressing different miR-27a levels confirmed the protein variations and recapitulated the cell growth and apoptosis effects. In vivo miR-27a inversely correlated with MHC class I molecules and calreticulin expression, CD8(+) T cells infiltration and cytotoxic activity (LAMP-1 exposure and perforin release). Tumours with high miR-27a, low calreticulin and CD8(+) T cells' infiltration were associated with distant metastasis and poor prognosis. Our data demonstrate that miR-27a acts as an oncomiRNA, represses MHC class I expression through calreticulin downregulation and affects tumour progression. These results may pave the way for better diagnosis, patient stratification and novel therapeutic approaches.
Purpose
Sabarubicin (MEN 10755), a new disaccaride anthracycline, has shown greater efficacy than Doxorubicin in a large panel of preclinical models and now it is in phase II clinical trials. Its ...promising antitumour activity promoted considerable interest to combine Sabarubicin with other antitumour agents. Thus, the purpose of this study was to evaluate in vitro cytotoxic effects and in vivo antitumour activities produced by the combination of Sabarubicin and cisplatin (DDP).
Methods
The antitumour effect of Sabarubicin and DDP association was investigated, in vitro and in vivo, in preclinical models of lung cancer i.e.: the non-small cell lung carcinoma (NSCLC) H460 and the small-cell lung carcinoma (SCLC) GLC4 in terms of synergism, additivity or antagonism in order to establish the best schedule for the combined treatment. Further, the correlation between antitumour activity and the pharmacokinetic parameters of the studied combination was also evaluated.
Results
The drug combination in vitro was in general more cytotoxic than the single drug alone, indicating the presence of a synergistic effect in both tumour cell lines. Also, in the xenograft experiments a superior antitumoral effect was observed when Sabarubicin was combined with DDP. The antitumour efficacy of Sabarubicin (6 mg/kg q4d × 5) combined with DDP (6 mg/kg q4d × 3) greatly depended on the schedule of administration. In H460 tumour line, the sequential combination was more effective than the simultaneous administration of the two agents, although the antitumour efficacy was not dependent on the sequence of combination. On the other hand, a strong sequence-dependent effect was observed when Sabarubicin was combined with DDP in SCLC, GLC4. In particular, the highest value of LCK = 6.7 was obtained when administration of DDP followed by 24 h that of Sabarubicin. Pharmacokinetics of Sabarubicin in combination with DDP was evaluated at 6 mg/kg for both drugs with different sequential schedule. The experimental data showed no evidence for pharmacokinetics drug–drug interaction.
Conclusion
These preclinical results indicate the potential for a strong antitumour activity in lung tumours of the combination Sabarubicin and DDP. In particular, in SCLC the best response should be given by a sequence with administration of Sabarubicin followed 24 h later by that of DDP. Clinical trials based on these results are ongoing.
The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. Mutations in PIK3CA, the gene coding the p110α catalytic subunit of PI3K, are ...frequent in cancer and result in PI3K pathway activation. About 25% of HER2-positive breast cancers also carry PIK3CA mutations, known to confer resistance to anti-HER2 therapy. Here we report the preclinical characterization of MEN1611, a potent and selective orally available PI3K inhibitor, showing high activity against p110α mutant isoforms and sparing the δ isoform.
In vivo efficacy studies were evaluated through Tumour Volume Inhibition % (TVI%) and mRECIST criteria using cancer cell lines and patient-derived xenograft (PDX) models. Pathway activation and protein degradation analyses were performed by western blot and capillary electrophoresis immunoassay. Immune modulation was evaluated using murine macrophages differentiated in vitro.
Consistent with its mechanism of action, MEN1611 down-modulates the PI3K/AKT signaling as well as selected downstream targets, both in vitro and in vivo. MEN1611 acts synergistically when combined with trastuzumab in several HER2-positive PIK3CA-mutated breast cancer cell lines and patient-derived xenograft models. The in vivo efficacy in trastuzumab-resistant models is supported by a long-lasting antitumor activity, paralleled by mechanistic down-regulation of pharmacodynamic biomarkers. Moreover, MEN1611 showed the ability to induce a dose-dependent alpha-isoform depletion, and to modulate the macrophage polarization towards a pro-inflammatory phenotype, consistent with its capability to co-inhibit P110γ.
Overall, these preclinical data support the progression in the development of MEN1611 in breast cancer and pave the way to the B-Precise-01 clinical study, a multicentre phase Ib study.
NCT03767335.
Menarini Ricerche S.p.A.
Menarini Ricerche S.p.A.
A. Fiascarelli: Full / Part-time employment: Menarini RIcerche S.p.A. G. Merlino: Full / Part-time employment: Menarini Ricerche S.p.A. S. Capano: Full / Part-time employment: Menarini Ricerche S.p.A. A. Paoli: Full / Part-time employment: Menarini Ricerche S.p.A.. A. Bressan: Full / Part-time employment: Menarini Ricerche. M. Bigioni: Full / Part-time employment: Menarini Ricerche S.p.A.. M. Scaltriti: Advisory / Consultancy: Memorial Sloan-Kettering Cancer Center. J. Arribas: Advisory / Consultancy: Vall d’Hebron Institute of Oncology. C. Bernadó Morales: Advisory / Consultancy: Vall d’Hebron Institute of Oncology. A. Pellacani: Leadership role, Full / Part-time employment: Menarini Ricerche S.p.A.. M. Salerno: Leadership role, Full / Part-time employment: Menarini Ricerche. M. Binaschi: Leadership role, Full / Part-time employment: Menarini Ricerche S.p.A.
Several observations highlight the importance of the carbohydrate moiety for the biological activity of antitumoural anthracyclines. Here is reported the synthesis, cytotoxicity and topoisomerase ...II-mediated DNA cleavage intensity of the new oligosaccharide anthracyclines 1--4 modified in the sugar residue. Evaluation of cytotoxic potency on different cell lines, resulted in quite similar values among the different analogues. On the other hand, topoisomerase II-mediated DNA breaks level was different for the various compounds, and was not related to cytotoxicity, thus supporting previous observations reported for some monosaccharide anthracyclines modified in the carbohydrate portion.
Topoisomerase II (top2) has been implicated in the initial steps of chromosomal translocations leading to leukemias and lymphomas, since it can generate DNA cleavage. To evaluate the effects of ...chromatin structure on enzyme-mediated cleavage, we determined the kinetics of loss of double-stranded DNA breaks stimulated by top2 poisons in Drosophila melanogaster Kc cells at two genomic regions that differ in chromatin structure. Moreover, cleavage loss was determined at 25 degrees C as well as after heat shock. Kinetics were dependent on the poison, nevertheless, loss rate overall was slow at the histone gene cluster, an active chromatin domain. At the repressed satellite III DNA, loss of cleavage was much faster and complete after 5 min in drug-free medium. In addition, differences were noted among sites that were closely spaced and equally intense. Following heat shock at 37 degrees C, we observed reduced cleavage levels and faster loss of breaks at the histone gene cluster. In vitro reversal could only partially explain the in vivo kinetics. Thus, the chromatin context of DNA breaks might play a role in the loss of top2 DNA breaks. The present findings suggest that irreversible cuts may more likely occur in active than silent loci.
Chemical agents able to interfere with DNA topoisomerases are widespread in nature, and some of them have clinical efficacy as antitumor or antibacterial drugs. Drugs which have as a target DNA ...topoisomerases could be divided into two categories: poisons and catalytic inhibitors. Classical topoisomerase poisons stimulate cleavage in a sequence-selective manner, yielding drug-specific cleavage intensity pattern. The mechanisms of drug interaction with DNA topoisomerases, the DNA sequence selectivity of the action of topoisomerase II poisons and the identification of structural determinants of their activity have suggested that topoisomerase II poisons may fit into a specific pharmacophore, constituted by a planar ring system with DNA intercalation or intercalation-like properties, and protruding side chains interfering with the protein side of the covalent enzyme–DNA complex. The complete definition of the diverse pharmacophores of topoisomerase II poisons will certainly be of value for the design of new agents directed to specific genomic sites, and more effective in the treatment of human cancer.
To better define the role of the amino sugar in the pharmacological and biochemical properties of anthracyclines related to doxorubicin and daunorubicin, we have investigated the effects of various ...substituents at the 3'- and 4'-positions of the drug on cytotoxic activity and ability to stimulate DNA cleavage mediated by DNA topoisomerase II. The study shows that the nature of the substituent at the 3'-position but not the 4'-position is critical for drug ability to form cleavable complexes. The amino group at the 3'-position is not essential for cytotoxic and topoisomerase II-targeting activities, because it can be replaced by a hydroxyl group without reduction of activity. However, the presence of bulky substituents at this position (i.e., morpholinyl derivatives) totally inhibited the effects on the enzyme, thus supporting previous observations indicating that the cytotoxic potencies of these particular derivatives are not related to topoisomerase II inhibition. This conclusion is also supported by the observation that 3'-morpholinyl and 3'-methoxymorpholinyl derivatives are able to overcome atypical (i.e., topoisomerase II-mediated) multidrug resistance. Because a bulky substituent at the 4'-position did not reduce the ability to stimulate DNA cleavage, these results support a critical role of the 3'-position in the drug interaction with topoisomerase II in the ternary complex. An analysis of patterns of cross-resistance to the studied derivatives in resistant human tumor cell lines expressing different resistance mechanisms indicated that chemical modifications at the 3'-position of the sugar may have a relevant influence on the ability of the drugs to overcome specific mechanisms of resistance.
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