[Retracted] Resistance to the mTOR inhibitor everolimus is reversed by the downregulation of survivin in breast cancer cells.

[This retracts the article DOI: 10.3892/ol.2017.6597.].

Design, Synthesis and Biological Evaluation of New Pyrimidine Derivatives as Anticancer Agents.

BACKGROUND: Anticancer drug resistance is a challenging phenomenon of growing concern which arises from alteration in drug targets. Despite the fast speed of new chemotherapeutic agent design, the increasing prevalence of this phenomenon requires further research and treatment development. Recently, we reported a new aminopyrimidine compound-namely RDS 344-as a potential innovative anticancer agent. METHODS: Herein, we report the design, synthesis, and anti-proliferative activity of new aminopyrimidine derivatives structurally related to RDS 3442 obtained by carrying out substitutions at position 6 of the pyrimidine core and/or on the 2-aniline ring of our hit. The ability to inhibit cell proliferation was evaluated on different types of tumors, glioblastoma, triple-negative breast cancer, oral squamous cell carcinomas and colon cancer plus on human dermal fibroblasts chosen as control of normal cells. RESULTS: The most interesting compound was the N-benzyl counterpart of RDS 3442, namely 2a, that induced a significant decrease in cell viability in all the tested tumor cell lines, with EC50s ranging from 4 and 8 µM, 4-13 times more active of hit. CONCLUSIONS: These data suggest a potential role for this class of molecules as promising tool for new approaches in treating cancers of different histotype.

Tissue Expression of Androgen Receptor Splice Variant 7 at Radical Prostatectomy Predicts Risk of Progression in Untreated Nonmetastatic Prostate Cancer.

BACKGROUND: Androgen receptor splice variant V7 (AR-V7) was recently detected in circulating tumor cells of castration-resistant prostate cancer (PC) patients and its expression correlated with resistance to new-generation androgen signaling inhibitors. OBJECTIVES: We retrospectively analyzed whether AR-V7 expression was detectable on radical prostatectomy (RP) specimens of untreated nonmetastatic PC cases, and whether it could be associated with progression after surgery. METHOD: The expression of AR-V7 and AR-FL (full length) was separately evaluated by immunohistochemistry using a streptavidin-biotin-peroxidase system with 2 anti-AR-V7 and anti-AR-FL rabbit monoclonal antibodies. RESULTS: 56 PC cases, classified by their clinical risk, were analyzed. Positive expression was found in 24/32 cases in the high-risk group, 4/13 in the intermediate-risk group, and only 2/11 in the low-risk group. We found a significant correlation between AR-V7 positivity and both risk classification (p < 0.001) and progression after surgery (p < 0.001). CONCLUSIONS: In our population of untreated nonmetastatic PC, AR-V7 is detectable by immunohistochemistry in more than 50% of cases. At this early stage, AR-V7 positivity is associated with risk classification and it can predict progression after surgery.

Discovery of a pyrimidine compound endowed with antitumor activity.

Recently, some synthetic nitrogen-based heterocyclic molecules, such as PJ34, have shown pronounced antitumor activity. Therefore, we designed and synthesized new derivatives characterized by a nitrogen-containing scaffold and evaluated their antiproliferative properties in tumor cells. We herein report the effects of three newly synthesized compounds on cell lines from three different human cancers: triple-negative breast cancer, colon carcinoma and glioblastoma. We found that two of these compounds did not affect proliferation, while the third significantly inhibited replication of the three cell lines. Moreover, this third molecule at 20 µM led to the upregulation of p21 and p27 and blockage of the cell cycle at G0/G1; in addition, it induced apoptosis in all three cell lines when used at higher concentrations (30-50 µM). The results demonstrate that this compound is a potent inhibitor of replication, an inducer of apoptosis and a negative regulator of cell cycle progression for cancer cells of different histotypes. Our data suggest a potential role for this new molecule as an interesting and powerful tool for new approaches in treating various cancers.

Dissecting the role of novel EZH2 inhibitors in primary glioblastoma cell cultures: effects on proliferation, epithelial-mesenchymal transition, migration, and on the pro-inflammatory phenotype.

BACKGROUND: Glioblastoma (GBM) is the most lethal and aggressive malignant primary brain tumor in adults. After surgical resection of the tumor, the patient typically should be subjected to chemotherapy (temozolomide, TMZ) and concomitant radiotherapy. Since the TMZ treatment does not lead to complete remission and often develops resistance, the identification of efficacious therapeutics is strongly to pursue. Among the epigenetic players, the H3K27 methyltransferase (MT) EZH2 (enhancer of zeste homologue 2) has been found overexpressed or mutated in several human cancers including gliomas, and its overexpression is associated with poor outcome in GBM. Two EZH2 inhibitors (EZH2i), UNC1999 and GSK343, suppressed GBM growth in vitro and in vivo indicating that EZH2i can be potential drugs against GBM. RESULTS: Two new EZH2i, MC4040 and MC4041, were designed, prepared, and tested by us to determine their effects in primary GBM cell cultures. MC4040 and MC4041 displayed single-digit micromolar inhibition of EZH2, 10-fold less potency against EZH1, and no activity towards other MTs. In primary GBM cells as well as in U-87 GBM cells, the two compounds reduced H3K27me3 levels, and dose- and time-dependently impaired GBM cell viability without inducing apoptosis and arresting the cell cycle in the G0/G1 phase, with increased p21 and p27 levels. In combination with TMZ, MC4040 and MC4041 displayed stronger, but not additive, effects on cell viability. The potent clinical candidate as EZH2i tazemetostat, alone or in combination with TMZ, exhibited a similar potency of inhibition of GBM cell growth when compared to MC4040 and MC4041. At the molecular level, MC4040 and MC4041 reduced the VEGFR1/VEGF expression, reversed the epithelial-mesenchymal transition (EMT), and hampered cell migration and invasion attenuating the cancer malignant phenotype. Treatment of GBM cells with MC4040 and MC4041 also impaired the GBM pro-inflammatory phenotype, with a significant decrease of TGF-ß, TNF-α, and IL-6, joined to an increase of the anti-inflammatory cytokine IL-10. CONCLUSIONS: The two novel EZH2i MC4040 and MC4041 impaired primary GBM cell viability, showing even stronger effects in combination with TMZ. They also weakened the aggressive malignant phenotype by reducing angiogenesis, EMT, cell migration/invasion and inflammation, thus they may be considered potential candidates against GBM also for combination therapies.

Growth arrest and apoptosis induced by kinesin Eg5 inhibitor K858 and by its 1,3,4-thiadiazoline analogue in tumor cells.

Tumors are complex and heterogeneous but, despite this, they share the ability to proliferate continuously, irrespective of the presence of growth signals, leading to a higher fraction of actively growing and dividing cells compared with normal tissues. For this reason, the cytotoxic antimitotic treatments remain an important clinical tool for tumors. Among these drugs, antitubulin compounds constitute one of the most effective anticancer chemotherapies; however, they cause dose-limiting side effects. Therefore, it is still necessary to develop compounds with new targets and new mechanisms of action to reduce side effects or chemoresistance. Mitosis-specific kinesin Eg5 can represent an attractive target for discovering such new anticancer agents because its role is fundamental in mitotic progression. Therefore, we analyzed the effects induced by an inhibitor of kinesin Eg5, K858, and by its 1,3,4-thiadiazoline analogue on human melanoma and prostate cancer cell lines. We found that both compounds have an antiproliferative effect, induce apoptosis, and can determine a downmodulation of survivin.

The kinesin Eg5 inhibitor K858 induces apoptosis and reverses the malignant invasive phenotype in human glioblastoma cells.

Glioblastoma multiforme is the most common primary malignant brain tumor and its current chemotherapeutic options are limited to temozolomide. Recently, some synthetic compounds acting as inhibitors of kinesin spindle protein Eg5 have shown pronounced antitumor activity. Our group has recently demonstrated that one of these kinesin Eg5 inhibitors, named K858, exerted important antiproliferative and apoptotic effects on breast cancer cells. Since glioblastoma cells usually express high levels of kinesin Eg5, we tested the effect of K858 on two human glioblastoma cell lines (U-251 and U-87) and found that K858 inhibited cell growth, induced apoptosis, reversed epithelial-mesenchymal transition and inhibited migration in both cell lines. We also detected that, at the same time, K858 increased the expression of survivin, an anti-apoptotic molecule, and that the forced down-regulation of survivin, obtained with the specific inhibitor YM155, boosted K858-dependent apoptosis. This indicated that the anti-tumor activity of K858 on glioblastoma cells is limited by the over-expression of survivin and that the negative regulation of this protein sensitizes tumor cells to K858. These data confirmed that kinesin Eg5 is an interesting target for new therapeutic approaches for glioblastoma. We showed that K858, specifically, was a potent inhibitor of replication, an inducer of apoptosis and a negative regulator of the invasive phenotype for glioblastoma cells.

Thyroid hormone regulates fibronectin expression through the activation of the hypoxia inducible factor 1.

Thyroid hormones regulate gene expression via both canonical and non-canonical signaling. Hyperthyroidism is associated with elevated plasma levels of fibronectin (FN): in this study we elucidate the molecular mechanism through which triiodothyronine (T3) regulates FN and demonstrate that T3 induces FN expression via a non-canonical pathway by activating hypoxia-inducible factor-1 (HIF-1). We found that T3 treatment increased cellular and secreted FN in human hepatoma cells (HepG2) and human dermal fibroblasts (HF) via the PI3K/Akt/HIF-1 pathway. The inhibition of either Akt phosphorylation with wortmannin or HIF-1 with YC1 in both cell types prevented HIF-1α synthesis and FN positive regulation upon T3 treatment. We showed that HIF-1α overexpression per se was sufficient to up-regulate FN in both cell lines as demonstrated by the transient transfection of both the constitutively active and wild-type forms of HIF-1α. Our data demonstrate the involvement of the PI3K/Akt/HIF-1 pathway in mediating T3 induced FN up-regulation.

Resistance to the mTOR inhibitor everolimus is reversed by the downregulation of survivin in breast cancer cells.

Everolimus (RAD001) is an inhibitor of mammalian target of rapamycin used in combination with exemestane to treat hormone receptor-positive advanced breast cancer. However, not all patients are equally sensitive to RAD001 and certain patients develop resistance. Therefore, the present study analyzed the mechanisms involved in the resistance of breast cancer cells to RAD001 in order to identify a potential tool to overcome it. The effects of RAD001 on the inhibition of cell viability, on the induction of apoptosis and autophagy and on the regulation of survivin, an anti-apoptotic protein, were evaluated in two breast cancer cell lines: BT474 (luminal B) and MCF7 (luminal A). RAD001 was demonstrated to induce autophagy in the two cell lines at following a short period of treatment (4 h) and to induce apoptosis exclusively in BT474 cells following longer periods of treatment (48 h). RAD001 induced the downregulation of survivin in BT474 cells and its upregulation in MCF7 cells. Consequently, inhibiting survivin with YM155 resulted in the acquired resistance of MCF7 cells to RAD001 being reverted, restoring RAD001-induced apoptosis. These data demonstrated that RAD001 exerted anti-proliferative and pro-apoptotic effects on breast cancer cells, but that these effects were repressed by the simultaneous up-regulation of survivin. Finally, the results demonstrated that inhibiting the expression of survivin resulted in the restoration of the anti-neoplastic activity of RAD001.

Circulating neuregulin-1 and galectin-3 can be prognostic markers in breast cancer.

BACKGROUND: It is important to identify novel plasmatic biomarkers that can contribute to assessing the prognosis and outcome of breast cancer patients. Neuregulin-1 (NRG1) and galectin-3 (Gal-3) are proteins that are involved in breast cancer development and patient survival; therefore, we studied whether the serum concentration of these 2 proteins can be correlated to breast cancer progression. METHODS: Plasmatic NRG1 and Gal-3 were evaluated in 25 healthy controls and 50 breast cancer patients at baseline and at 3 and 6 months after treatment with anthracyclines and taxanes, with or without trastuzumab. RESULTS: NRG1 and Gal-3 were significantly more elevated in cancer patients than in healthy controls; furthermore, NRG1 and Gal-3 were significantly increased after chemotherapy and were predictive of mortality at 1 year. CONCLUSIONS: Circulating NRG1 and Gal-3 can be additional biomarkers indicative of prognosis and outcomes for breast cancer patients.

Anthracycline-Free Neoadjuvant Chemotherapy Ensures Higher Rates of Pathologic Complete Response in Breast Cancer.

PURPOSE: Neoadjuvant chemotherapy (NCT) is a standard of care for locally advanced and initially inoperable breast cancer. NCT can test chemotherapy efficacy and can be followed by breast-conserving surgery. Considering taxanes as one of the most effective agents, we analyzed the efficacy of a neoadjuvant schedule without anthracyclines and based only on taxanes and carboplatin, trying to avoid cardiotoxicity, which is the most serious side effect correlated with anthracyclines. PATIENTS AND METHODS: We enrolled 61 patients with breast cancer, belonging to 4 subgroups, according to molecular phenotypes: 24 triple-negative/basal-like, 13 HER2-like, 20 luminal B, and 4 luminal A. All patients underwent weekly chemotherapy with carboplatin AUC2, paclitaxel 80 mg/m2, with trastuzumab (in case of HER2 positivity) 2 mg/kg, except for luminal A patients, who underwent only hormonal therapy. Among 61 patients, 26 (43%) received modified radical mastectomy and 35 (57%) received breast-conserving surgery. RESULTS: The patients obtaining pathologic complete response (pCR) were 20 (83%) of 24 triple-negative/basal-like, 10 (76%) of 13 HER2-like, 6 (30%) of 20 luminal B, and 3 (75%) of 4 luminal A. All the patients were evaluated for toxicity: no grade 4 was detected, 5 patients experienced grade 3 neuropathy, then reverted to G2 after chemotherapy discontinuation. At a minimum follow-up of 5 years, median overall survival was 48 months. CONCLUSION: Taxane/carboplatin-based/anthracycline-free NCT is the best treatment for inoperable breast cancer in terms of efficacy and toxicity, because this approach avoids cardiotoxicity and obtains an optimal rate (64%) of pCR, with an important impact on survival.

The kinesin Eg5 inhibitor K858 induces apoptosis but also survivin-related chemoresistance in breast cancer cells.

Inhibitors of kinesin spindle protein Eg5 are characterized by pronounced antitumor activity. Our group has recently synthesized and screened a library of 1,3,4-thiadiazoline analogues with the pharmacophoric structure of K858, an Eg5 inhibitor. We herein report the effects of K858 on four different breast cancer cell lines: MCF7 (luminal A), BT474 (luminal B), SKBR3 (HER2 like) and MDA-MB231 (basal like). We demonstrated that K858 displayed anti-proliferative activity on every analyzed breast cancer cell line by inducing apoptosis. However, at the same time, we showed that K858 up-regulated survivin, an anti-apoptotic molecule. We then performed a negative regulation of survivin expression, with the utilization of wortmannin, an AKT inhibitor, and obtained a significant increase of K858-dependent apoptosis. These data demonstrate that K858 is a potent inhibitor of replication and induces apoptosis in breast tumor cells, independently from the tumor phenotype. This anti-proliferative response of tumor cells to K858 can be limited by the contemporaneous over-expression of survivin; consequently, the reduction of survivin levels, obtained with AKT inhibitors, can sensitize tumor cells to K858-induced apoptosis.

Breast cancer cells respond differently to docetaxel depending on their phenotype and on survivin upregulation.

Breast cancer is characterized by molecular heterogeneity, and four major breast cancer subtypes have been identified, each characterized by significant differences in survival, prognosis, and response to therapy. We have studied the effects of docetaxel treatment on apoptosis and survivin expression in four breast cancer cell lines: MCF7 (luminal A: estrogen receptor-positive and progesterone receptor-positive, ErbB2-negative), BT474 (luminal B: estrogen receptor/progesterone receptor/ErbB2-positive), SKBR3 (HER2-like: estrogen receptor/progesterone receptor-negative, ErbB2-positive), and MDA-MB231 (basal-like: estrogen receptor/progesterone receptor/ErbB2-negative). We demonstrated that docetaxel-induced apoptosis and survivin upregulation (MCF7 p = 0.002, BT474 p = 0.001, SKBR3 p = 0.001) in luminal A/B and HER2-like cells, while it induced mainly necrosis and a lower rate of survivin upregulation (MDA-MB231 p = 0.035) in basal-like cells. Wortmannin, a p-Akt inhibitor, was able to revert surviving upregulation and, at the same time, induced an increase of docetaxel-dependent apoptosis, suggesting that reduced levels of survivin can sensitize tumor cells to apoptosis. These data show that the analyzed breast cancer cell lines respond differently to docetaxel, depending on their receptor expression profile and molecular phenotype. Yet, these data confirm that one of the pathways involved in taxane-related chemoresistance is the upregulation of survivin. Further studies on the molecular mechanisms of chemoresistance and on the different modalities of apoptosis induced by chemotherapeutic agents are requested to better understand how cancer cells evade cell death, in order to design new kind of anticancer agents and survivin could represent a future target for this kind of research.

Serum biomarkers evaluation to predict chemotherapy-induced cardiotoxicity in breast cancer patients.

Anti-neoplastic chemotherapy can determine various side effects, including cardiotoxicity, and no real guidelines for its early detection and management have been developed. The aim of this study is to find some plasmatic markers able to identify breast cancer patients that are at greater risk of developing cardiovascular complications during chemotherapy, in particular heart failure. A prospective study on 100 breast cancer patients with mean age of 66 years in adjuvant treatment with anthracyclines, taxanes, and trastuzumab was performed. Patients underwent cardiological examination before starting treatment (T0) and at 3 months (T1), 6 months (T2), and 1 year (T3) after treatment. Evaluation of serum cardiac markers and N-terminal pro-brain natriuretic peptide (NT-proBNP) was performed at T0, T1, T2, and T3, simultaneously to electrocardiogram and echocardiogram, showing a significant increase in NT-proBNP concentration (p > 0.0001) at T1, T2, and T3, before left ventricular ejection fraction decrease became evident. Human epidermal growth factor receptor 2 (HER2)-negative patients were more susceptible to mild hematological cardiotoxicity, while HER2-positive patients were more susceptible to severe cardiotoxicity. A significant correlation between NT-proBNP increased values after chemotherapy and prediction of mortality at 1 year was evidenced. From our experience, serum biomarker detection was able to support an early diagnosis of cardiac damage, also in the absence of left ventricular ejection fraction decrease. Therefore, the evaluation of specific plasmatic markers for cardiac damage is more sensitive than echocardiography in the early diagnosis of chemotherapy-related cardiotoxicity; furthermore, it can also add a prognostic value on outcome.

Are pharmacogenomic biomarkers an effective tool to predict taxane toxicity and outcome in breast cancer patients? Literature review.

PURPOSE: Breast cancer is a heterogeneous disease, characterized by various molecular phenotypes that correlate with different prognosis and response to treatments. Taxanes are some of the most active chemotherapeutic agents for breast cancer; however, their utilization is limited, due to hematologic and cumulative neurotoxicity on treated patients. To understand why only some patients experience severe adverse effects and why patients respond and develop resistance with different rates to taxane therapy, the metabolic pathways of these drugs should be completely unraveled. The variant forms of several genes, related to taxane pharmacokinetics, can be indicative markers of clinical parameters, such as toxicity or outcome. METHODS: The search of the data has been conducted through PubMed database, presenting clinical data, clinical trials and basic research restricted to English language until June 2015. RESULTS: We studied the literature in order to find any possible association between the major pharmacogenomic variants and specific taxane-related toxicity and patient outcome. We found that the data of these studies are sometimes discordant, due to both the small number of enrolled patients and the heterogeneity of the examined population. CONCLUSIONS: Among all analyzed genes, only CYP1B1 and ABCB1 resulted the strongest candidates to become biomarkers of clinical response to taxane therapy in breast cancer, although their utilization still remains an experimental procedure. In the future, greater studies on genetic polymorphisms should be performed in order to identify differentiating signatures for patients with higher toxicity and with resistant or responsive outcome, before the administration of taxanes.

Taxane induced neuropathy in patients affected by breast cancer: Literature review.

Taxane induced neuropathy (TIN) is the most limiting side effect of taxane based chemotherapy, relative to the majority of breast cancer patients undergoing therapy with both docetaxel and paclitaxel. The symptoms begin symmetrically from the toes, because the tips of the longest nerves are affected for first. The patients report sensory symptoms such as paresthesia, dysesthesia, numbness, electric shock-like sensation, motor impairment and neuropathic pain. There is a great inter-individual variability among breast cancer women treated with taxanes, in fact 20-30% of them don't develop neurotoxicity. Actually, there is no standard therapy for TIN, although many medications, antioxidants and natural substances have been tested in vitro and in vivo. We will summarize all most recent literature data on TIN prevention and treatment, in order to reach an improvement in TIN management. Further studies are needed to evaluate new therapies that restore neuronal function and improve life quality of patients.

On and off metronomic oral vinorelbine in elderly women with advanced breast cancer.

BACKGROUND: Elderly patients with metastatic breast cancer (MBC) have more problems receiving chemotherapy than younger patients, especially with the presence of multiple comorbidities, adverse drug events and functional decline. Low-dose oral administration of cytotoxic agents such as vinorelbine, a semisynthetic vinca alkaloid that interferes with microtubule assembly, leading to arrest of cell division, is usually effective and well tolerated. METHODS: From February 2010 to February 2014, 32 patients with MBC, median age 76 years (range 69-83) were treated with oral vinorelbine 30 mg (total dose), one day on and one day off, until disease progression or unacceptable toxicity levels were reported. Toxicity, quality of life and clinical benefit were evaluated. Matched t-tests were conducted to discern whether quality-of-life indicator (p<0.05 was considered significant) differed before and 6 months after treatment. Statistical analysis was performed using Graph Pad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). RESULTS: No grade 3 and 4 adverse events were reported. A clinical benefit of 50% was found in our cohort. On and off metronomic vinorelbine oral administration resulted in good tolerability and safe profile in our selected elderly population, and improved patient adherence to therapy. CONCLUSIONS: The present study demonstrated that metronomic vinorelbine might be a potential treatment in elderly patients by reducing adverse effects and increasing quality of life, setting the stage for future extensive clinical trials.

Chemotherapy of rare skin adnexal tumors: a review of literature.

Malignant skin adnexal tumors are rare neoplasms which are derived from adnexal epithelial structures of the skin: hair follicle, or sebaceous, apocrine or eccrine glands. Among them, eccrine porocarcinoma is the most frequent, with an aggressive behavior compared to other more common forms of non-melanoma skin cancer. Only few reports describe the treatment of metastatic adnexal tumors, and there is no consensus about the better strategy of chemotherapy. Given the few cases and the absence of randomized clinical trials, it is important to collect clinical experiences on these tumors. Most of these adenocarcinomas are very aggressive and also chemoresistant, and only a targeted-therapy could have an impact on patient survival. Therefore, further studies on the biology of these diseases are necessary. The purpose of the present review is to discuss the treatment of malignant neoplasms of cutaneous adnexae and to suggest some future therapeutic options based on targeted-therapy.