The AKT1E17K Allele Promotes Breast Cancer in Mice.

The gain-of-function mutation in the pleckstrin homology domain of AKT1 (AKT1E17K) occurs in lung and breast cancer. Through the use of human cellular models and of a AKT1E17K transgenic Cre-inducible murine strain (R26-AKT1E17K mice), we have demonstrated that AKT1E17K is a bona fide oncogene for lung epithelial cells. However, the role of AKT1E17K in breast cancer remains to be determined. Here, we report the generation and the characterization of a MMTV-CRE; R26-AKT1E17K mouse strain that expresses the mutant AKT1E17K allele in the mammary epithelium. We observed that AKT1E17K stimulates the development of mammary tumors classified as ductal adenocarcinoma of medium-high grade and presented a variety of proliferative alterations classified as adenosis with low-to-high grade dysplasia in the mammary epithelium. A subsequent immunohistochemical characterization suggested they were PR-/HER2-/ER+, basal-like and CK8-/CK10-/CK5+/CK14+. We also observed that, in parallel with an increased proliferation rate, tumors expressing mutant AKT1E17K presented an activation of the GSK3/cyclin D1 pathway in the mammary epithelium and cluster significantly with the human basal-like tumors. In conclusion, we demonstrate AKT1E17K is a bona fide oncogene that can initiate tumors at high efficiency in murine mammary epithelium in vivo.

Antitumor activity of novel POLA1-HDAC11 dual inhibitors.

Hybrid molecules targeting simultaneously DNA polymerase α (POLA1) and histone deacetylases (HDACs) were designed and synthesized to exploit a potential synergy of action. Among a library of screened molecules, MIR002 and GEM144 showed antiproliferative activity at nanomolar concentrations on a panel of human solid and haematological cancer cell lines. In vitro functional assays confirmed that these molecules inhibited POLA1 primer extension activity, as well as HDAC11. Molecular docking studies also supported these findings. Mechanistically, MIR002 and GEM144 induced acetylation of p53, activation of p21, G1/S cell cycle arrest, and apoptosis. Oral administration of these inhibitors confirmed their antitumor activity in in vivo models. In human non-small cancer cell (H460) xenografted in nude mice MIR002 at 50 mg/kg, Bid (qd × 5 × 3w) inhibited tumor growth (TGI = 61%). More interestingly, in POLA1 inhibitor resistant cells (H460-R9A), the in vivo combination of MIR002 with cisplatin showed an additive antitumor effect with complete disappearance of tumor masses in two animals at the end of the treatment. Moreover, in two human orthotopic malignant pleural mesothelioma xenografts (MM473 and MM487), oral treatments with MIR002 and GEM144 confirmed their significant antitumor activity (TGI = 72-77%). Consistently with recent results that have shown an inverse correlation between POLA1 expression and type I interferon levels, MIR002 significantly upregulated interferon-α in immunocompetent mice.

Novel adamantyl retinoid-related molecules with POLA1 inhibitory activity.

Atypical retinoids (AR) or retinoid-related molecules (RRMs) represent a promising class of antitumor compounds. Among AR, E-3-(3'-adamantan-1-yl-4'-hydroxybiphenyl-4-yl)acrylic acid (adarotene), has been extensively investigated. In the present work we report the results of our efforts to develop new adarotene-related atypical retinoids endowed also with POLA1 inhibitory activity. The effects of the synthesized compounds on cell growth were determined on a panel of human and hematological cancer cell lines. The most promising compounds showed antitumor activity against several tumor histotypes and increased cytotoxic activity against an adarotene-resistant cell line, compared to the parent molecule. The antitumor activity of a selected compound was evaluated on HT-29 human colon carcinoma and human mesothelioma (MM487) xenografts. Particularly significant was the in vivo activity of the compound as a single agent compared to adarotene and cisplatin, against pleural mesothelioma MM487. No reduction of mice body weight was observed, thus suggesting a higher tolerability with respect to the parent compound adarotene.

A Herbal Mixture from Propolis, Pomegranate, and Grape Pomace Endowed with Anti-Inflammatory Activity in an In Vivo Rheumatoid Arthritis Model.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by the production of inflammatory factors. In order to overcome the side effects of currently used anti-inflammatory drugs, several attempts have been made to identify natural products capable of relieving RA symptoms. In this work, a herbal preparation consisting of propolis, pomegranate peel, and Aglianico grape pomace (PPP) extracts (4:1:1) was designed and evaluated for its effect on a murine collagen-induced arthritis (CIA) model. Firstly, the chemical contents of four different Italian propolis collected in the Campania region (Italy) were here reported for the first time. LC-MS analyses showed the presence of 38 constituents, identified in all propolis extracts, belonging to flavonoids and phenolic acids classes. The Pietradefusi extract was the richest one and thus was selected to design the PPP preparation for the in vivo assay. Our results highlight the impact of PPP on RA onset and progression. By using in vivo CIA models, the treatment with PPP resulted in a delayed onset of the disease and alleviated the severity of the clinical symptoms. Furthermore, we demonstrated that early PPP treatment was associated with a reduction in serum levels of IL-17, IL-1b, and IL-17-triggering cytokines.

Hybrid topoisomerase I and HDAC inhibitors as dual action anticancer agents.

Recent studies have shown that HDAC inhibitors act synergistically with camptothecin derivatives in combination therapies. To exploit this synergy, new hybrid molecules targeting simultaneously topoisomerase I and HDAC were designed. In particular, a selected multivalent agent containing a camptothecin and a SAHA-like template showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Camptothecin-psammaplin A hybrids as topoisomerase I and HDAC dual-action inhibitors.

Recent studies have demonstrated enhanced anticancer effects of combination therapy consisting of camptothecin derivatives and HDAC inhibitors. To exploit this synergy in a single active compound, we designed new dual-acting multivalent molecules simultaneously targeting topoisomerase I and HDAC. In particular, a selected compound containing a camptothecin and the psammaplin A scaffold showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Tumor response of temozolomide in combination with morphine in a xenograft model of human glioblastoma.

Despite multimodal treatments comprising, radiation therapy (RT) and chemotherapy with temozolomide (TMZ), the prognosis of glioblastoma multiforme (GBM) remains dismal and consolidated therapy yields a median survival of 14.6 months. Blood Brain Barrier (BBB) mediated chemoresistance and high dose related toxicity make necessary the development of new therapeutic approach to sensitize GBM to TMZ. The aim of the present study was to investigate the potential of the treatment morphine plus TMZ metronmic doses (1,77 and 0,9 mg/kg) in GBM therapy. The effect of morphine, on tumor cell growth and P-glycoprothein (P-gp) activity, was investigate in in vitro models. The results demonstrated that GBM cells growth is not influenced by morphine treatment and, for the first time, we show that morphine is an inhibitor of the activity of P-gp efflux transporter who is markedly expressed on BBB. In vivo, response to the treatments TMZ plus morphine was investigated in an orthotopic nude mice model of GBM. Animals treated with TMZ metronomic doses showed a significant tumor growth inhibition compared to untreated mice and association with morphine appears to improve TMZ efficacy. Moreover, the combination of morphine with lower dose of TMZ result in a cytostatic effect on tumor growth over the period of the pharmacological treatments. In conclusion this novel approach could be a successful strategy to overcome chemoresistance and side effects TMZ mediated, reducing drug dosage and improving long term response, in GBM therapy.

AKT1E¹7K Is Oncogenic in Mouse Lung and Cooperates with Chemical Carcinogens in Inducing Lung Cancer.

The hotspot AKT1E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6-2% of human lung cancers. Recently, we have demonstrated that AKT1E17K transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1E17K mice) we demonstrate that AKT1E17K is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1E17K induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1E17K induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype.

The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells.

Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC.