Giulio Francia

VEGF-A polymorphisms predict progression-free survival among advanced castration-resistant prostate cancer patients treated with metronomic cyclophosphamide.

Background:No data are available on the pharmacogenetics of metronomic chemotherapy in prostate cancer. The aim of this study was to evaluate the association between VEGF-A sequence variants and prostate-specific antigen (PSA) progression, progression-free survival (PFS) and overall survival (OS), in advanced castration-resistant prostate cancer patients treated with metronomic cyclophosphamide (CTX), celecoxib and dexamethasone.Methods:Forty-three patients were enrolled, and genomic DNA was extracted. VEGF-A gene SNPs (−2578A/C, −634C/G, +936C/T) were analysed using TaqMan PCR assays. Hardy–Weinberg equilibrium was tested for each SNP, and genetic effects were evaluated by Fisher’s exact test. PFS and OS were analysed with GraphPad Prism software, using the product limit method of Kaplan and Meier, and comparing survival curves using both the log-rank test and the Gehan–Wilcoxon test. We used Bonferroni correction to account for multiple testing, and a two-tailed P-value of <0.017 was considered statistically significant.Results:Overall, 20 patients (46%) experienced a reduction in PSA levels from baseline and, among them, 14 (32%) showed a confirmed PSA ≥50% decrease. In non-responders, the −2578CC genotype was more frequent (18.60% vs 2.33% in responders; P=0.0212) whereas the −634CC genotype frequency was 22.73% vs 0% in responders (P=0.0485). With regard to PFS, patients harbouring the −634CC genotype had a median PFS of 2.2 months whereas patients with the genotype −634CG/GG had a median PFS of 6.25 months (P=0.0042).Conclusion:The −634CC genotype is significantly associated with a shorter PFS in patients treated with a metronomic CTX schedule.

Preclinical recapitulation of antiangiogenic drug clinical efficacies using models of early or late stage breast cancer metastatis

Historically, preclinical tumor therapy models in mice have frequently been deficient in predicting subsequent clinical activity; over-prediction of clinical anti-tumor efficacy is common. Several approaches are being made in an attempt to improve the clinical relevance of preclinical models, and include the use of genetically engineered mouse models (GEMMs) of cancer or patient derived xenografts (PDXs). Here we summarize, in the context of breast cancer, another approach, namely, the development of postsurgical models of either macroscopic or microscopic metastatic disease to mimic metastatic or adjuvant therapy. To do so we used in vivo selected metastatic variants of established human breast cancer cell lines such as MDA-MB-231. Testing antiangiogenic drugs such as the oral tyrosine kinase inhibitor (TKI) sunitinib alone or combined with chemotherapy in models involving treatment of established primary tumors invariably resulted in demonstrable anti-tumor activity. In contrast, identical treatments of postsurgical mice with advanced metastatic disease did not: survival times were not prolonged. This reflects multiple failed phase III trials of sunitinib based therapies in metastatic breast cancer patients. However, using a VEGF pathway targeting antibody drug instead of a TKI, with (paclitaxel) chemotherapy, resulted in anti-tumor activity in the metastatic setting, partially reflecting prior clinical results of the E2100 phase III trial of weekly paclitaxel plus bevacizumab. Other experiments involving postsurgical adjuvant treatment of early stage disease foreshadowed the phase III clinical trial failures of adjuvant bevacizumab in colorectal or breast cancer. In contrast, some investigational metronomic oral chemotherapy protocols alone or in combination with an antiangiogenic drug demonstrated potent activity in the advanced metastatic setting; these encouraging results have yet to be validated in randomized phase III clinical trials, which are underway based on some encouraging phase II clinical trial results. We have also observed circumstances where mice with advanced systemic disease, when successfully treated so as to prolong survival, sometimes relapse with brain metastases, reflecting a similar clinical phenomenon. Given our overall findings, we suggest that using preclinical mouse tumor models which mimic postsurgical adjuvant or metastatic therapy may be a promising strategy to help improve the ability to predict subsequent clinical outcomes.

A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells

PurposeAccording to the World Health Organization (WHO), breast cancer is the most common cancer affecting women worldwide. In the USA ~12.3xa0% of all women are expected to be diagnosed with various types of breast cancer, exhibiting varying degrees of therapeutic response rates. Therefore, the identification of novel anti-breast cancer drugs is of paramount importance.MethodsThe 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore was incorporated into a number of cytotoxins. Three of the resulting dienones, 2a, 2b and 2c, were tested for their anti-neoplastic potencies in a variety of human breast cancer-derived cell lines, including the triple negative MDA-MB-231 cell line and its metastatic variant, using a live-cell bio-imaging method. Special emphasis was put on dienone 2c, since its anti-cancer activity and its mode of inflicting cell death have so far not been reported.ResultsWe found that all three dienones exhibited potent cytotoxicities towards the breast cancer-derived cell lines tested, whereas significantly lower toxicities were observed towards the non-cancerous human breast cell line MCF-10A. The dienones 2b and 2c exhibited the greatest selective cytotoxicity at submicromolar concentration levels. We found that these two dienones induced phosphatidylserine externalization in MDA-MB-231 cells in a concentration-dependent manner, suggesting that their cytotoxic effect might be mediated by apoptosis. This possibility was confirmed by our observation that the dienone 2c can induce mitochondrial depolarization, caspase-3 activation, cell cycle disruption and DNA fragmentation in MDA-MB-231 cells.ConclusionOur findings indicate that dienone 2c uses the mitochondrial/intrinsic pathway to inflict apoptosis in triple negative MDA-MB-231 breast cancer-derived cells. This observation warrants further assessment of dienone 2c as a potential anti-breast cancer drug.

Docetaxel plus oral metronomic cyclophosphamide: A phase II study with pharmacodynamic and pharmacogenetic analyses in castration-resistant prostate cancer patients

Docetaxel plus prednisone is currently the standard first‐line treatment in metastatic castration‐resistant prostate cancer (mCRPC). The aim of this study was to assess the clinical activity and pharmacodynamic/pharmacogenetic profile of docetaxel plus prednisone in combination with metronomic cyclophosphamide in mCRPC patients.

Preclinical analysis of resistance and cross-resistance to low-dose metronomic chemotherapy

SummaryLow-dose metronomic chemotherapy is an emerging form of chemotherapy with distinct mechanisms of action from conventional chemotherapy (e.g., antiangiogenesis). Although developed to overcome resistance to conventional chemotherapy, metronomic chemotherapy is subject to resistance on its own. However, there is a paucity of information on mechanisms of resistance, on cross-resistance between metronomic regimens using different cytotoxic drugs, and on cross-resistance between metronomic versus conventional chemotherapy, or versus targeted antiangiogenic therapy. Herein we show that PC-3 human prostate cancer xenografts were sensitive to both metronomic cyclophosphamide and metronomic docetaxel, but resistant to metronomic topotecan. Conventional docetaxel was only moderately active in parental PC-3 and in metronomic cyclophosphamide resistant PC-3 tumors. However, in metronomic cyclophosphamide resistant PC-3 tumors combining conventional docetaxel or bolus cyclophosphamide therapy with continued metronomic cyclophosphamide was superior to each treatment alone. Furthermore, bevacizumab had single-agent activity against metronomic cyclophosphamide resistant PC-3 tumors. Microarray analyses identified altered regulation of protein translation as a potential mechanism of resistance to metronomic cyclophosphamide. Our results suggest that sensitivity to metronomic chemotherapy regimens using different cytotoxic drugs not only depends on shared mechanisms of action such as antiangiogenesis, but also on as yet unknown additional antitumor effects that appear to be drug-specific. As clinically observed with targeted antiangiogenic agents, the continued use of metronomic chemotherapy beyond progression may amplify the effects of added second-line therapies or vice versa. However, metronomic chemotherapy is no different from other systemic therapies in that predictive biomarkers will be essential to fully exploit this novel use of conventional chemotherapeutics.

Antiproliferative and Proapoptotic Activity of Sunitinib on Endothelial and Anaplastic Thyroid Cancer Cells via Inhibition of Akt and ERK1/2 Phosphorylation and by Down-Regulation of Cyclin-D1.

CONTEXTnRecent experimental evidence suggests a rationale for the use of multitarget tyrosine kinase inhibitors for the treatment of thyroid cancers. Sunitinib showed promising preliminary results against anaplastic thyroid cancer (ATC), and it has been used for some patients who are ineligible for clinical trials.nnnOBJECTIVESnThe aims of this study were to investigate the in vitro and in vivo activity of sunitinib on ATC and on microvascular endothelial cells and the molecular mechanism for the observed sunitinib activity.nnnMETHODSnProliferation and apoptotic assays were performed on human dermal microvascular endothelial and on BRAF- or H-ras-mutated ATC cells (8305C and FB3, respectively) after in vitro exposure to sunitinib for 72 hours. Vascular endothelial growth factor receptor-2, epithelial growth factor receptor, ERK1/2, and Akt phosphorylation was quantified by ELISA and Western blot. Cyclin-D1 mRNA expression was evaluated by real-time PCR, and cyclin-D1 intracellular concentrations were measured by ELISA. 8305C tumor xenografts in nude mice were treated with sunitinib at 50 mg/kg/d (i.p.).nnnRESULTSnAntiproliferative and proapoptotic activity of sunitinib was observed in both endothelial and ATC cells. Phospho-vascular endothelial growth factor receptor-2 levels significantly decreased after sunitinib treatment in activated endothelial cells. Phospho-epidermal growth factor receptor, ERK1/2, and Akt phosphorylation was significantly inhibited by sunitinib treatment in endothelial and cancer cells, and cyclin-D1 mRNA and protein expression was inhibited. Sunitinib administration in vivo caused significant inhibition of tumor growth (P < .05).nnnCONCLUSIONSnSunitinib is active in vitro and in vivo against activated endothelial and ATC cells via the inhibition of Akt and ERK1/2 phosphorylation and through the down-regulation of cyclin-D1.

p62/IMP2 stimulates cell migration and reduces cell adhesion in breast cancer

p62/IMP2 is an oncofetal protein that is overexpressed in several types of cancer, and is a member of the family of insulin-like growth factor 2 mRNA binding proteins. We previously reported that high levels of p62/IMP2 autoantibody are present in sera from cancer patients, compared to healthy individuals. Here, we report the overexpression of p62/IMP2 in tumor tissues of 72 out of 104 cases of human breast cancer, and high levels of p62/IMP2 autoantibody in patients’ sera (in 63 out of 216 cases). To explore the role of p62/IMP2 in breast cancer progression, we generated p62/IMP2 transfected variants of two human breast cancer cell lines: MDA-MB-231 and LM2-4. Using in vitro assays we found that overexpression of p62/IMP2 can increase cell migration, and reduce cell adhesion to extracellular matrix (ECM) proteins. A Human Extracellular Matrix and Adhesion Molecules qPCR array was performed with our generated variants, and it identified a group of mRNAs whose expression was altered with p62/IMP2 overexpression, including connective tissue growth factor (CTGF) mRNA – which we show to be a p62/IMP2 binding partner. Overall, our results provide new insights into the molecular mechanism by which p62/IMP2 can contribute to breast cancer progression.

Metronomic vinorelbine is directly active on Non Small Cell Lung Cancer cells and sensitizes the EGFRL858R/T790M cells to reversible EGFR tyrosine kinase inhibitors

Graphical abstract Figure. No Caption available. ABSTRACT Metronomic vinorelbine (mVNR) has been described primarily as an antiangiogenic therapy, and no direct effects of mVNR on Non Small Cell Lung Cancer (NSCLC) cells has yet been demonstrated. The aims of this study were i) to establish the direct activity of mVNR on NSCLC cells either EGFR wt or EGFRL858R/T790M, and ii) to quantify the synergism of the combination with reversible EGFR tyrosine kinase inhibitors (TKIs), investigating the underlying mechanism of action. Proliferation assays were performed on A‐549 (wt EGFRhigh), H‐292 (EGFR‐wt), H‐358 (EGFR‐wt), H‐1975 (EGFRL858R/T790M) NSCLC cell lines exposed to mVNR, its active metabolite deacetyl‐VNR (D‐VNR), gefitinib and erlotinib for 144 h treatments. The synergism between mVNR and EGFR TKIs was determined by the combination index (CI) in EGFR‐wt and H‐1975 NSCLC cells. Cyclin‐D1 and ABCG2 genes expression and protein levels were measured by RT‐PCR and ELISA assays, as well as the phosphorylation of ERK1/2 and Akt. Intracellular concentrations of EGFR TKIs and VNR were investigated with a mass spectrometry system. mVNR, and its active metabolite D‐VNR, were extremely active on NSCLC cells, in particular on H‐1975 (IC50 = 13.56 ± 2.77 pM), resistant to TKIs. mVNR inhibited the phosphorylation of ERK1/2 and Akt and significantly decreased the expression of both cyclin‐D1 and ABCG2 m‐RNA and protein. The simultaneous combination of VNR and reversible EGFR TKIs showed a strong synergism on EGFR‐wt NSCLC cells and on H‐1975 cells (e.g. CI = 0.501 for 50% of affected cells), increasing the intracellular concentrations of EGFR TKIs (e.g. +50.5% vs. gefitinib alone). In conclusions, mVNR has direct effects on NSCLC cells and sensitizes resistant cells to EGFR TKIs, increasing their intracellular concentrations.

Clinical, pharmacodynamic and pharmacokinetic results of a prospective phase II study on oral metronomic vinorelbine and dexamethasone in castration-resistant prostate cancer patients.

SummaryThe aim of the present study was to evaluate clinical activity, and the pharmacodynamic and pharmacokinetic profiles, of oral metronomic vinorelbine (VNR) plus dexamethasone (DEX) in metastatic castration-resistant prostate cancer (mCRPC) patients. Fourty-one patients (92xa0% chemotherapy-resistant) received 30xa0mg/day VNR p.o. thrice a week plus 1xa0mg/day DEX p.o. until disease progression. Plasma soluble B cell antigen 7 homolog 3 (sB7-H3), vascular endothelial growth factor (VEGF), and thrombospondin-1 (TSP-1), were measured by ELISA. Plasma VNR was detected using a LC-MS-MS system. The fraction of patients free of progression, defined by criteria of the Prostate Cancer Clinical Trials Working Group 2, at 3xa0months was 61xa0%. PSA decrease ≥50xa0% from baseline was observed in 35xa0% of patients. Median PFS and OS were 4xa0months (95xa0% CI, 2.8–6.9) and 17.5xa0months (95xa0% CI, 10.8–24.5), respectively. Toxicity was mild, and no grade 4 toxicities were found. The mean plasma VNR Cmax ranged from 1 to 2.7xa0ng/ml (Tmax 1.1xa0h) and no evidence of drug accumulation was found. A moderate relationship was found between plasma sB7-H3 and PSA values (rxa0=xa00.565; Pxa0=xa00.0094) at the baseline. Increased PFS (11.3 vs. 2.8xa0months; Pxa0=xa00.0298) was observed in patients with sB7-H3 levels <30.25xa0ng/mL. Plasma VEGF AUC0-24day increased in non-responders (Pxa0<xa00.0001), whereas responders maintained higher plasma TSP-1 AUC0-24day (Pxa0=xa00.0063). In conclusion, metronomic VNR plus DEX showed favourable activity, and a low toxicity profile, in mCRPC patients. Plasma sB7-H3, VEGF and TSP-1 levels are potential pharmacodynamic markers at the reached low plasma concentrations of vinorelbine metronomically administered.

Resistance to metronomic chemotherapy and ways to overcome it

Therapeutic resistance is amongst the major determinants of cancer mortality. Contrary to initial expectations, antivascular therapies are equally prone to inherent or acquired resistance as other cancer treatment modalities. However, studies into resistance to vascular endothelial growth factor pathway inhibitors revealed distinct mechanisms of resistance compared to conventional cytotoxic therapy. While some of these novel mechanisms of resistance also appear to be functional regarding metronomic chemotherapy, herein we summarize available evidence for mechanisms of resistance specifically described in the context of metronomic chemotherapy. Numerous preclinically identified molecular targets and pathways represent promising avenues to overcome resistance and enhance the benefits achieved with metronomic chemotherapy eventually. However, there are considerable challenges to clinically translate the preclinical findings.