The kinetochore protein Cenp‐F is a potential novel target for zoledronic acid in breast cancer cells

The anti‐resorptive agent zoledronic acid inhibits key enzymes in the mevalonate pathway, disrupting post‐translational modification and thereby correct protein localization and function. Inhibition of prenylation may also be responsible for the reported anti‐tumour effects of zoledronic acid, but the specific molecular targets have not been identified. Cenp‐F/mitosin, a kinetochore‐associated protein involved in the correct separation of chromosomes during mitosis, has been shown to undergo post‐translational prenylation and may therefore be a novel target contributing to the anti‐tumour effects of zoledronic acid. We investigated whether zoledronic acid causes loss of Cenp‐F from the kinetochore in breast cancer cells, to determine if the reported anti‐tumour effects may be mediated by impairing correct chromosome separation. MDA‐MB‐436, MDA‐MB‐231 and MCF‐7 breast cancer cells and MCF‐10A non‐malignant breast epithelial cells were treated with zoledronic acid in vitro, and the effect on Cenp‐F localization was analysed by immunoflourescence microscopy. Zoledronic acid caused loss of Cenp‐F from the kinetochore, accompanied by an increase in the number of cells in pro‐, /prometa‐ and metaphase in all of the cancer cell lines. There was also a significant increase in the number of lagging chromosomes in mitotic cells. The effects of zoledronic acid could be reversed by inclusion of an intermediary of the mevalonate pathway, showing that the loss of Cenp‐F from the kinetochore was caused by the inhibition of farnesylation. In contrast, no effect was seen on Cenp‐F in non‐malignant MCF‐10A cells. This is the first report showing a specific effect of zoledronic acid on a protein involved in the regulation of chromosome segregation, identifying Cenp‐F as a potential new molecular target for NBPs in tumour cells.     

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.     

Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway

Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition.     

Tumour-released exosomes and their implications in cancer immunity

Tumour cells release vesicular structures, defined as microvesicles or exosomes, carrying a large array of proteins from their originating cell. The expression of antigenic molecules recognized by T cells has originally suggested a role for these organelles as a cell-free antigen source for anticancer vaccines. However, recent evidence shows that tumour exosomes may also exert a broad array of detrimental effects on the immune system, ranging from apoptosis in activated antitumour T cells to impairment of monocyte differentiation into dendritic cells and induction of myeloid suppressive cells. Immunosuppressive exosomes of tumour origin can be found in neoplastic lesions and sera from cancer patients, implying a potential role of this pathway in in vivo tumour progression. Through the expression of molecules involved in angiogenesis promotion, stromal remodelling, delivery of signalling pathways through growth factor/receptor transfer, chemoresistance and genetic intercellular exchange, tumour exosomes could represent a versatile tool for moulding host environment. Hence, their secretion by neoplastic cells may in the future become a novel pathway to target for therapeutic intervention in cancer patients.     

Radiation combines with immune checkpoint blockade to enhance T cell priming in a murine model of poorly immunogenic pancreatic cancer

Radiation has been a pillar of cancer therapy for decades. The effects of radiation on the anti-tumour immune response are variable across studies and have not been explicitly defined in poorly immunogenic tumour types. Here, we employed combination checkpoint blockade immunotherapy with stereotactic body radiation therapy and examined the effect on tumour growth and immune infiltrates in subcutaneous and orthotopic mouse models of pancreatic cancer. Although immune checkpoint blockade and radiation were ineffective alone, their combination produced a modest growth delay in both irradiated and non-irradiated tumours that corresponded with significant increases in CD8+ T cells, CD4+ T cells and tumour-specific T cells as identified by IFNγ ELISpot. We conclude that radiation enhances priming of tumour-specific T cells in poorly immunogenic tumours and that the frequency of these T cells can be further increased by combination with immune checkpoint blockade.     

Mevalonate promotes the growth of tumors derived from human cancer cells in vivo and stimulates proliferation in vitro with enhanced cyclin-dependent kinase-2 activity

Malignant cells are known to have elevated rates of mevalonate synthesis because of increased levels and catalytic efficiency of 3-hydroxy-3-methylglutaryl-CoA reductase. Whether this increased mevalonate synthesis occurs as a consequence of increased requirements for a mevalonate-derived metabolite in response to rapid growth or whether mevalonate promotes the growth of tumor cells is unknown. To address this question, we administered mevalonate via miniosmotic pumps to nude mice inoculated with MDA-MB-435 human cancer cells. After 13 weeks of growth, tumors in mevalonate-treated mice were significantly larger than tumors in saline-treated, control mice (1.52 +/- 0.26 g versus 0.81 +/- 0.27 g respectively, p < 0.05). The cancer cells treated in culture with mevalonate also demonstrated increased proliferation rates associated with accelerated entry of cells into S phase. These cells had enhanced total and cyclin A-immunoprecipitable cyclin-dependent kinase-2 (CDK-2) activity, increased activating phosphorylation of CDK-2, and decreased inhibitory binding of CDK-2 to p21(Cip1). Our findings demonstrate that mevalonate promotes tumor growth and suggest that an increase in mevalonate synthesis in extrahepatic tissues following cholesterol-lowering therapy may explain the elevated risk of cancer shown in some studies.     

Lysosomal-Mitochondrial Axis in Zoledronic Acid-induced Apoptosis in Human Follicular Lymphoma Cells

Bisphosphonates (BPs) are potent inhibitors of osteoclast function, widely used to treat excessive bone resorption associated with bone metastases, that also have anti-tumor activity. Zoledronic acid (ZOL) represents a potential chemotherapeutic agent for the treatment of cancer. ZOL is the most potent nitrogen-containing BPs, and it inhibits cell growth and induces apoptosis in a variety of cancer cells. Recently we demonstrated that accumulation of isopentenyl pyrophosphate and the consequent formation of a new type of ATP analog (ApppI) after mevalonate pathway inhibition by nitrogen-containing BPs strongly correlates with ZOL-induced cell death in cancer cells in vitro. In this study we show that ZOL-induced apoptosis in HF28RA human follicular lymphoma cells occurs exclusively via the mitochondrial pathway, involves lysosomes, and is dependent on mevalonate pathway inhibition. To define the exact signaling pathway connecting them, we used modified HF28RA cell lines overexpressing either BclXL or dominant-negative caspase-9. In both mutant cells, mitochondrial and lysosomal membrane permeabilization (MMP and LMP) were totally prevented, indicating signaling between lysosomes and mitochondria and, additionally, an amplification loop for MMP and/or LMP regulated by caspase-9 in association with farnesyl pyrophosphate synthetase inhibition. Additionally, the lysosomal pathway in ZOL-induced apoptosis plays an additional/amplification role of the intrinsic pathway independently of caspase-3 activation. Moreover, we show a potential regulation by Bcl-XL and caspase-9 on cell cycle regulators of S-phase. Our findings provide a molecular basis for new strategies concomitantly targeting cell death pathways from multiple sites.     

Cancer stem cells: the theory and perspectives in cancer therapy

The cancer stem cell theory elucidates not only the issue of tumour initiation and development, tumour's ability to metastasise and reoccur, but also the ineffectiveness of conventional cancer therapy. This review examines stem cell properties, such as self-renewal, heterogeneity, and resistance to apoptosis. The 'niche' hypothesis is presented, and mechanisms of division, differentiation, self-renewal and signalling pathway regulation are explained. Epigenetic alterations and mutations of genes responsible for signal transmission may promote the formation of cancer stem cells. We also present the history of development of the cancer stem cell theory and discuss the experiments that led to the discovery and confirmation of the existence of cancer stem cells. Potential clinical applications are also considered, including therapeutic models aimed at selective elimination of cancer stem cells or induction of their proper differentiation.     

Oncometabolites in cancer aggressiveness and tumour repopulation

Tumour repopulation is recognized as a crucial event in tumour relapse where therapy‐sensitive dying cancer cells influence the tumour microenvironment to sustain therapy‐resistant cancer cell growth. Recent studies highlight the role of the oncometabolites succinate, fumarate, and 2‐hydroxyglutarate in the aggressiveness of cancer cells and in the worsening of the patient's clinical outcome. These oncometabolites can be produced and secreted by cancer and/or surrounding cells, modifying the tumour microenvironment and sustaining an invasive neoplastic phenotype. In this review, we report recent findings concerning the role in cancer development of succinate, fumarate, and 2‐hydroxyglutarate and the regulation of their related enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase. We propose that oncometabolites are crucially involved in tumour repopulation. The study of the mechanisms underlying the relationship between oncometabolites and tumour repopulation is fundamental for identifying efficient anti‐cancer therapeutic strategies and novel serum biomarkers in order to overcome cancer relapse.     

Immune modulation by dendritic-cell-based cancer vaccines

The interplay between host immunity and tumour cells has opened the possibility of targeting tumour cells by modulation of the human immune system. Cancer immunotherapy involves the treatment of a tumour by utilizing the recombinant human immune system components to target the pro-tumour microenvironment or by revitalizing the immune system with the ability to kill tumour cells by priming the immune cells with tumour antigens. In this review, current immunotherapy approaches to cancer with special focus on dendritic cell (DC)-based cancer vaccines are discussed. Some of the DC-based vaccines under clinical trials for various cancer types are highlighted. Establishing tumour immunity involves a plethora of immune components and pathways; hence, combining chemotherapy, radiation therapy and various arms of immunotherapy, after analysing the benefits of individual therapeutic agents, might be beneficial to the patient.     

Cholesterol and beyond - The role of the mevalonate pathway in cancer biology

Cancer is a multifaceted global disease. Transformation of a normal to a malignant cell takes several steps, including somatic mutations, epigenetic alterations, metabolic reprogramming and loss of cell growth control. Recently, the mevalonate pathway has emerged as a crucial regulator of tumor biology and a potential therapeutic target. This pathway controls cholesterol production and posttranslational modifications of Rho-GTPases, both of which are linked to several key steps of tumor progression. Inhibitors of the mevalonate pathway induce pleiotropic antitumor-effects in several human malignancies, identifying the pathway as an attractive candidate for novel therapies. In this review, we will provide an overview about the role and regulation of the mevalonate pathway in certain aspects of cancer initiation and progression and its potential for therapeutic intervention in oncology.     

Coagulation and inflammation in cancer: Limitations and prospects for treatment

The development of so-called immune checkpoint inhibitors (ICIs), which target specific molecular processes of tumour growth, has had a transformative effect on cancer treatment. Widespread use of antibody-based medicines to inhibit tumour cell immune evasion by modulating T cell responses is becoming more common. Despite this, response rates are still low, and secondary resistance is an issue that arises often. In addition, a wide range of serious adverse effects is triggered by enhancing the immunological response. As a result of an increased mortality rate, a higher prevalence of thrombotic complications is connected with an increased incidence of immunological reactions, complement activation, and skin toxicity. This suggests that the tumour microenvironment's interaction between coagulation and inflammation is important at every stage of the tumour's life cycle. The coagulation system's function in tumour formation is the topic of this review. By better understanding the molecular mechanisms in which tumour cells circulate, plasmatic coagulation and immune system cells are engaged, new therapy options for cancer sufferers may be discovered.     

Effect of phytosterols on cholesterol metabolism and MAP kinase in MDA-MB-231 human breast cancer cells

Epidemiological studies suggest that dietary phytosterols may offer protection form some types of cancer including breast cancer. In an attempt to investigate the mechanism by which phytosterols offer this protection, we investigated the effect of the two most common dietary phytosterols, beta-sitosterol and campesterol, on the mevalonate and MAP Kinase (MAPK) pathways in MDA-MB-231 cells. These pathways play a role in cell growth and apoptosis. MDA-MB-231 cell line was used in this study since it is a hormone-insensitive tumor cell line which represents the majority of advanced breast cancer cases. Cells grown in the presence of 16 microM beta-sitosterol or campesterol for 3 days exhibited a 70% and 6% reduction in cell growth, respectively, while cholesterol treatment had no effect on growth as compared to the control. Studies investigating the effect of sterol supplementation on the relative and total sterol composition of cells, showed that cells supplemented with cholesterol contained 23% more cholesterol than the control. Cells supplemented with campesterol had almost one-half the cholesterol of controls but accumulated campesterol to account for 40% of the total sterols. In the case of cells supplemented with beta-sitosterol, cells had only 25% of their sterols as cholesterol and the rest was in the form of beta-sitosterol. All sterols tested equally inhibited de novo cholesterol synthesis using 14C-acetate as substrate. beta-Sitosterol supplemented cells had reduced cholesterol synthesis when using 3H-mevalonolactone as substrate, which suggests that the inhibition in this pathway is downstream of mevalonate where processes such as isoprenylation of proteins may take place. Mevalonate supplementation to cells treated with beta-sitosterol did not completely correct the observed growth inhibition by beta-sitosterol. There was no effect of sterols on the concentrations of both low (21-26 kDa) or high (44-74 kDa) molecular weight isoprenylated proteins in these cells. On the other hand, both the quantity and activity of MAPK was elevated in the cells supplemented with beta-sitosterol. These data suggest that the down regulation of cholesterol synthesis from mevalonate and stimulation of the MAPK pathway may play roles in the inhibition of MDA-MB-231 cell growth by beta-sitosterol.     

Lung cancer and Toll-like receptors

Lung carcinoma is one of the leading causes of death worldwide. It is a non-immunogenic cancer, resistant to immune surveillance. Toll-like receptors (TLRs) connect the innate to the adaptive immune system. Given that cancerous cells evade the immune system, the activation of TLRs could represent a potential target for cancer therapy. The induction of Th1-like and cytotoxic immunity by TLR signalling could lead to tumour cell death, resulting in tumour regression or arrest. However, basic research and clinical trials revealed that the activation of specific TLRs, such as TLR2, TLR4 and TLR9, do not have any anti-tumour activity in lung carcinoma. Increasing evidence suggests that TLRs are important regulators of tumour biology; however, little is known about their function in lung cancer. Thus, in order to develop new therapeutic approaches, further studies are needed to understand the connection between TLRs and lung cancer progression. This review focuses on the potential mechanisms by which TLR ligands can facilitate or not lung cancer and lung metastases establishment/progression.     

Gene therapy of neoplastic liver diseases

Since advanced liver cancer lacks effective therapy in most cases, a considerable interest has been drawn towards gene therapy. Natural or chimerical genes can be transferred to the tumour itself, the non-tumoral liver, or even distant tissues using a variety of vectors administered by intratumoral or intravascular routes. The desired selectivity in gene expression can be achieved by increasing the specificity of gene delivery or by controlling gene expression with tumour-specific promoters, such as alpha-fetoprotein or carcinoembryonic antigen. There are two main approaches to gene therapy of liver cancer aiming at killing directly malignant cells or at improving the host's defensive systems, respectively. The former include replacing the lost function of tumour suppressor genes, inhibiting the action of activated oncogenes, sensitising tumour cells to prodrugs, or infecting the tumoral tissue with viruses that replicate selectively in cancer cells. Host defences can be improved by stimulating the antitumoral immune response, or by interfering with tumour vessel formation. Progress in gene therapy of liver cancer depends very much on information collected from well-designed clinical trials. This information includes knowledge of whether an efficient gene transfer has been achieved and what is the duration and magnitude of gene expression in the transduced tissues. Hopefully, magnetic resonance or positron emission tomography (PET) may turn out to be reliable procedures for tracing transgene expression in humans. Pre-clinical evidence and early clinical trials strongly suggest that there is a place for gene therapy of liver malignancies.     

TGFbeta in Cancer

The transforming growth factor beta (TGFbeta) signaling pathway is a key player in metazoan biology, and its misregulation can result in tumor development. The regulatory cytokine TGFbeta exerts tumor-suppressive effects that cancer cells must elude for malignant evolution. Yet, paradoxically, TGFbeta also modulates processes such as cell invasion, immune regulation, and microenvironment modification that cancer cells may exploit to their advantage. Consequently, the output of a TGFbeta response is highly contextual throughout development, across different tissues, and also in cancer. The mechanistic basis and clinical relevance of TGFbeta's role in cancer is becoming increasingly clear, paving the way for a better understanding of the complexity and therapeutic potential of this pathway.     

Inhibitory B7-family molecules in the tumour microenvironment

The B7 family consists of activating and inhibitory co-stimulatory molecules that positively and negatively regulate immune responses. Recent studies have shown that human and rodent cancer cells, and stromal cells and immune cells in the cancer microenvironment upregulate expression of inhibitory B7 molecules and that these contribute to tumour immune evasion. In this Review, we focus on the roles of these B7 molecules in the dynamic interactions between tumours and the host immune system, including their expression, regulation and function in the tumour microenvironment. We also discuss novel therapeutic strategies that target these inhibitory B7 molecules and their signalling pathways to treat human cancer.     

Tumour suppressor genes in chemotherapeutic drug response

Since cancer is one of the leading causes of death worldwide, there is an urgent need to find better treatments. Currently, the use of chemotherapeutics remains the predominant option for cancer therapy. However, one of the major obstacles for successful cancer therapy using these chemotherapeutics is that patients often do not respond or eventually develop resistance after initial treatment. Therefore identification of genes involved in chemotherapeutic response is critical for predicting tumour response and treating drug-resistant cancer patients. A group of genes commonly lost or inactivated are tumour suppressor genes, which can promote the initiation and progression of cancer through regulation of various biological processes such as cell proliferation, cell death and cell migration/invasion. Recently, mounting evidence suggests that these tumour suppressor genes also play a very important role in the response of cancers to a variety of chemotherapeutic drugs. In the present review, we will provide a comprehensive overview on how major tumour suppressor genes [Rb (retinoblastoma), p53 family, cyclin-dependent kinase inhibitors, BRCA1 (breast-cancer susceptibility gene 1), PTEN (phosphatase and tensin homologue deleted on chromosome 10), Hippo pathway, etc.] are involved in chemotherapeutic drug response and discuss their applications in predicting the clinical outcome of chemotherapy for cancer patients. We also propose that tumour suppressor genes are critical chemotherapeutic targets for the successful treatment of drug-resistant cancer patients in future applications.