Obesity, non-alcoholic fatty liver disease and hepatocellular carcinoma: current status and therapeutic targets.

Obesity is a global epidemic and overwhelming evidence indicates that it is a risk factor for numerous cancers, including hepatocellular carcinoma (HCC), the third leading cause of cancer-related deaths worldwide. Obesity-associated hepatic tumorigenesis develops from nonalcoholic fatty liver disease (NAFLD), progressing to nonalcoholic steatohepatitis (NASH), cirrhosis and ultimately to HCC. The rising incidence of obesity is resulting in an increased prevalence of NAFLD and NASH, and subsequently HCC. Obesity represents an increasingly important underlying etiology of HCC, in particular as the other leading causes of HCC such as hepatitis infection, are declining due to effective treatments and vaccines. In this review, we provide a comprehensive overview of the molecular mechanisms and cellular signaling pathways involved in the pathogenesis of obesity-associated HCC. We summarize the preclinical experimental animal models available to study the features of NAFLD/NASH/HCC, and the non-invasive methods to diagnose NAFLD, NASH and early-stage HCC. Finally, since HCC is an aggressive tumor with a 5-year survival of less than 20%, we will also discuss novel therapeutic targets for obesity-associated HCC and ongoing clinical trials.

The role of transcription factors in the acquisition of the four latest proposed hallmarks of cancer and corresponding enabling characteristics.

With the recent description of the molecular and cellular characteristics that enable acquisition of both core and new hallmarks of cancer, the consequences of transcription factor dysregulation in the hallmarks scheme has become increasingly evident. Dysregulation or mutation of transcription factors has long been recognized in the development of cancer where alterations in these key regulatory molecules can result in aberrant gene expression and consequential blockade of normal cellular differentiation. Here, we provide an up-to-date review of involvement of dysregulated transcription factor networks with the most recently reported cancer hallmarks and enabling characteristic properties. We present some illustrative examples of the impact of dysregulated transcription factors, specifically focusing on the characteristics of phenotypic plasticity, non-mutational epigenetic reprogramming, polymorphic microbiomes, and senescence. We also discuss how new insights into transcription factor dysregulation in cancer is contributing to addressing current therapeutic challenges.

A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line.

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and ß-glycerophosphate (ßG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to ßG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with ßG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.

The Cancer Stem Cell Niche in Ovarian Cancer and Its Impact on Immune Surveillance.

Ovarian cancer is an aggressive gynaecological cancer with extremely poor prognosis, due to late diagnosis as well as the development of chemoresistance after first-line therapy. Research advances have found stem-like cells present in ovarian tumours, which exist in a dynamic niche and persist through therapy. The stem cell niche interacts extensively with the immune and non-immune components of the tumour microenvironment. Significant pathways associated with the cancer stem cell niche have been identified which interfere with the immune component of the tumour microenvironment, leading to immune surveillance evasion, dysfunction and suppression. This review aims to summarise current evidence-based knowledge on the cancer stem cell niche within the ovarian cancer tumour microenvironment and its effect on immune surveillance. Furthermore, the review seeks to understand the clinical consequences of this dynamic interaction by highlighting current therapies which target these processes.

Calcium transport and signalling in breast cancer: Functional and prognostic significance.

Comprised of a complex network of numerous intertwining pathways, the Ca2+ signalling nexus is an essential mediator of many normal cellular activities. Like many other such functions, the normal physiological activity of Ca2+ signalling is frequently co-opted and reshaped in cases of breast cancer, creating a potent oncogenic drive within the affected cell population. Such modifications can occur within pathways mediating either Ca2+ import (e.g. TRP channels, ORAI-STIM1) or Ca2+ export (e.g. PMCA), indicating that both increases and decreases within cellular Ca2+ levels have the potential to increase the malignant potential of a cell. Increased understanding of these pathways may offer clinical benefit in terms of both prognosis and treatment; patient survival has been linked to expression levels of certain Ca2+ transport proteins, whilst selective targeting of these factors with novel anti-cancer agents has demonstrated a variety of anti-tumour effects in in vitro studies. In addition, the activity of several Ca2+ signalling pathways has been shown to influence chemotherapy response, suggesting that a synergistic approach coupling traditional chemotherapy with Ca2+ targeting agents may also improve patient outcome. As such, targeted modulation of these pathways represents a novel approach in precision medicine and breast cancer therapy.

Academic staff perspectives on delivering a shared undergraduate medical module on three transnational campuses: Practical considerations and lessons learned.

The Royal College of Surgeons in Ireland (RCSI) was among the first medical institutions to establish a global education community which now provides high-quality transnational health professions education aligned across three locations: Europe, the Middle East and South-East Asia. The successful implementation of a shared modularized curriculum in this context can be complex and challenging. Here we describe our insights, gained from a decade of working together as shared module Academic Leads to deliver a system-based medical module to an international student cohort. The themes covered are some of the areas where we consider our joint deliberations have led to improved outcomes for the delivery and assessment of the module, which may be helpful to academic staff embarking on similar module sharing experiences.

Deposition of calcium in an in vitro model of human breast tumour calcification reveals functional role for ALP activity, altered expression of osteogenic genes and dysregulation of the TRPM7 ion channel.

Microcalcifications are vital mammographic indicators contributing to the early detection of up to 50% of non-palpable tumours and may also be valuable as prognostic markers. However, the precise mechanism by which they form remains incompletely understood. Following development of an in vitro model using human breast cancer cells lines cultured with a combination of mineralisation-promoting reagents, analysis of calcium deposition, alkaline phosphatase (ALP) activity and changes in expression of key genes was used to monitor the calcification process. Two cell lines were identified as successfully mineralising in vitro, MDA-MB-231 and SKBR3. Mineralising cell lines displayed higher levels of ALP activity that was further increased by addition of mineralisation promoting media. qPCR analysis revealed changes in expression of both pro- (RUNX2) and anti- (MGP, ENPP1) mineralisation genes. Mineralisation was suppressed by chelation of intracellular Ca2+ and inhibition of TRPM7, demonstrating a functional role for the channel in formation of microcalcifications. Increased Mg2+ was also found to effectively reduce calcium deposition. These results expand the number of human breast cancer cell lines with a demonstrated in vitro mineralisation capability, provide further evidence for the role of an active, cellular process of microcalcification formation and demonstrate for the first time a role for TRPM7 mediated Ca2+ transport.

A novel platinum complex of the histone deacetylase inhibitor belinostat: rational design, development and in vitro cytotoxicity.

The successful design and synthesis of a novel Pt complex of the histone deacteylase inhibitor belinostat are reported. Molecular modelling assisted in the identification of a suitable malonate derivative of belinostat (mal-p-Bel) for complexation to platinum. Reaction of [Pt(NH3)2(H2O)2](NO3)2 with the disodium salt of mal-p-Bel gave cis-[Pt(NH3)2(mal-p-Bel-2H)] (where -2H indicates that mal-p-Bel is doubly deprotonated) in excellent yield. An in vitro cytotoxicity study revealed that cis-[Pt(NH3)2(mal-p-Bel-2H)] possesses (i) considerable cytotoxicity against reported ovarian cancer cell lines, (ii) enhanced cytotoxicity relative to the previously reported Pt histone deacetylase inhibitor conjugate, cis-[Pt(II)(NH3)2(malSAHA-2H)] and (iii) favourable cyto-selective properties as compared to cisplatin and belinostat.

Microcalcifications in breast cancer: Lessons from physiological mineralization.

Mammographic mammary microcalcifications are routinely used for the early detection of breast cancer, however the mechanisms by which they form remain unclear. Two species of mammary microcalcifications have been identified; calcium oxalate and hydroxyapatite. Calcium oxalate is mostly associated with benign lesions of the breast, whereas hydroxyapatite is associated with both benign and malignant tumors. The way in which hydroxyapatite forms within mammary tissue remains largely unexplored, however lessons can be learned from the process of physiological mineralization. Normal physiological mineralization by osteoblasts results in hydroxyapatite deposition in bone. This review brings together existing knowledge from the field of physiological mineralization and juxtaposes it with our current understanding of the genesis of mammary microcalcifications. As an increasing number of breast cancers are being detected in their non-palpable stage through mammographic microcalcifications, it is important that future studies investigate the underlying mechanisms of their formation in order to fully understand the significance of this unique early marker of breast cancer.

Osteomimicry of mammary adenocarcinoma cells in vitro; increased expression of bone matrix proteins and proliferation within a 3D collagen environment.

Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.

Novel trans-platinum complexes of the histone deacetylase inhibitor valproic acid; synthesis, in vitro cytotoxicity and mutagenicity.

The first examples of Pt complexes of the well known anti-epilepsy drug and histone deacetylase inhibitor, valproic acid (VPA), are reported. Reaction of the Pt(II) am(m)ine precursors trans-[PtCl(2)(NH(3))(py)] and trans-[PtCl(2)(py)(2)] with silver nitrate and subsequently sodium valproate gave trans-[Pt(VPA(-1H))(2)(NH(3))(py)] and trans-[Pt(VPA(-1H))(2)(py)(2)], respectively. The valproato ligands in both complexes are bound to the Pt(II) centres via the carboxylato functionality and in a monodentate manner. The X-ray crystal structure of trans-[Pt(VPA(-1H))(2)(NH(3))(py)] is described. Replacement of the dichlorido ligands in trans-[PtCl(2)(py)(2)] and trans-[PtCl(2)(NH(3))(py)] by valproato ligands (VPA(-1H)) to yield trans-[Pt(VPA(-1H))(2)(py)(2)] and trans-[Pt(VPA(-1H))(2)(NH(3))(py)] respectively, significantly enhanced cytotoxicity against A2780 (parental) and A2780 cisR (cisplatin resistant) ovarian cancer cells. The mutagenicity of trans-[Pt(VPA(-1H))(2)(NH(3))(py)] and trans-[Pt(VPA(-1H))(2)(py)(2)] was determined using the Ames test and is also reported.

A novel anti-cancer bifunctional platinum drug candidate with dual DNA binding and histone deacetylase inhibitory activity.

The successful design and synthesis of a novel multifunctional platinum drug candidate with DNA binding, histone deacetylase inhibitory activity and enhanced selectivity for cancer cells are described.

Microcalcifications associated with breast cancer: an epiphenomenon or biologically significant feature of selected tumors?

Radiographic mammary calcifications occur in 30-50% of breast cancers and constitute one of the most important diagnostic markers of both benign and malignant lesions of the breast. The presence of oxalate-type microcalcification appears to be a reliable criterion in favor of the benign nature of the lesion or, at most, of a lobular carcinoma in situ. In contrast, calcium hydroxyapatite (HA) crystals are associated with both benign and malignant breast tumors. Although the diagnostic value of microcalcifications in breast cancer is of great importance, the genesis of these calcifications is unclear. Despite numerous histological ultrastructure studies of HA deposits in breast carcinomas, to date there have been limited investigations of the potential role of these crystals in breast cancer. We review the literature examining the biological effects of HA crystals in breast cancer cell lines, specifically the mechanism of HA-induced mitogenesis and upregulation of gene expression.

Basic calcium phosphate crystals as a unique therapeutic target in osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis that occurs in humans. Despite its prevalence, the pathogenesis of OA is not fully understood. Intraarticular basic calcium phosphate (BCP) (an inclusive term for partially carbonate-substituted hydroxyapatite, octacalcium phosphate and tricalcium phosphate) crystals are implicated in OA and are associated with severe degenerative arthritis characterized by marked synovial hyperplasia, aggravated joint degeneration and large joint effusions. Their pathogenicity relates, at least in part, to their ability to stimulate cellular mitogenesis in a number of cell types including macrophages, porcine articular chondrocytes (PAC) and human fibroblasts (HF) and induce prostaglandin, cytokine and matrix metalloproteinase synthesis and secretion in HF and PAC. Identification of BCP crystals in OA joints remains problematic because of the lack of a simple and reliable analytic procedure. There is currently no drug available that prevents the formation or modifies the biological effects of BCP crystals. This review highlights the recent advances in our knowledge of BCP crystal deposition diseases and discusses the potential therapeutic strategies for BCP crystal-associated OA.

Basic calcium phosphate crystal-induced prostaglandin E2 production in human fibroblasts: role of cyclooxygenase 1, cyclooxygenase 2, and interleukin-1beta.

OBJECTIVE: To elucidate the mechanism of basic calcium phosphate (BCP) crystal-induced prostaglandin E(2) (PGE(2)) production in human foreskin fibroblasts (HFFs), to identify the signaling pathway involved in the induction of cyclooxygenase 2 (COX-2) messenger RNA (mRNA) by BCP crystals, to examine the effect of BCP crystals on interleukin-1beta (IL-1beta) mRNA expression, and to investigate the potential of phosphocitrate to abrogate the BCP crystal-induced effects. METHODS: PGE(2) levels were quantified using a commercial enzyme immunoassay kit. COX-2 and COX-1 transcript levels were quantified using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Induction of IL-1beta and COX-2 mRNA was examined by end-point RT-PCR. COX-2 protein expression was assessed by Western blotting. RESULTS: PGE(2) production measured 4 and 30 hours after BCP crystal treatment was higher in BCP crystal-treated (mean +/- SEM 1,891 +/- 273 pg/microg and 1,792 +/- 233 pg/microg, respectively) than in untreated (88 +/- 5 pg/microg and 205 +/- 93 pg/microg, respectively) HFFs. The PGE(2) produced after 4 hours was sensitive to inhibition with NS398, a selective COX-2 inhibitor, implying that it was COX-2 mediated, whereas the PGE(2) produced at 30 hours could not be completely inhibited by NS398. Real-time RT-PCR demonstrated a 23-fold increase in COX-2 mRNA that was maximal at 4 hours, whereas analysis of mRNA for COX-1 showed up-regulation of transcript peaking at 24 hours poststimulation (1.75-fold increase). The protein kinase C and phosphatidylinositol 3-kinase signal-transduction inhibitors bisindolylmaleimide I and LY294002, respectively, blocked BCP crystal-induced COX-2 mRNA in HFFs. In addition, BCP crystals were found to up-regulate the proinflammatory cytokine IL-1beta (maximal at 8 hours). The induction of both COX-2 and IL-1beta by BCP crystals was attenuated when the cells were treated with phosphocitrate. CONCLUSION: These findings indicate that BCP crystals may be an important amplifier of PGE(2) production through induction of the COX enzymes and the proinflammatory cytokine IL-1beta.

Phosphocitrate inhibits calcium hydroxyapatite induced mitogenesis and upregulation of matrix metalloproteinase-1, interleukin-1beta and cyclooxygenase-2 mRNA in human breast cancer cell lines.

Microcalcifications containing calcium hydroxyapatite (HA) are often associated with malignant human breast lesions. Frequently, they are the only mammographic features that indicate the presence of a tumoural lesion. We previously reported the induction of both mitogenesis and prostaglandin E2 (PGE2) production and the increased activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in normal human mammary epithelial cells and breast cancer cell lines, treated with HA. In the present study we attempted to elucidate the mechanism of these biological effects. Firstly, we found that direct cell-crystal contact was required for induction of mitogenesis as the effect was not merely a result of isotopic exchange of calcium into the culture medium. Treatment with bafilomycin A1, a proton pump inhibitor, abrogated HA-induced mitogenesis to control cell levels. These results suggest that phagocytosis and intracellular crystal dissolution is required for HA-induced mitogenesis. We also demonstrated that the increase in prostaglandin E2, previously reported, is due, at least in part, to HA-induced upregulation of cyclooxygenase-2 (COX-2) in Hs578T cells. An accumulation of MMP-1 mRNA was also shown in response to HA stimulation in Hs578T cells. Furthermore, a HA-induced increase in interleukin-1beta (IL-1beta), a potent inducer of MMP-1 gene expression, was demonstrated in Hs578T cells at 2 and 4 h. Treatment with phosphocitrate (PC) (a naturally occurring inhibitor of calcium phosphate crystallisation, which is known to block a number of HA-induced biological effects in other cell types) blocked HA-mediated mitogenesis, as well as, COX-2, MMP-1 and IL-1beta induction, at the transcriptional level. These results show that calcium HA crystals are capable of exerting significant biological effects on surrounding cells which can be abrogated by PC and emphasise the role of calcium HA in amplifying the pathological process involved in breast cancer.

Signaling mechanisms involved in crystal-induced tissue damage.

The association of crystal deposition with osteoarthritis and joint destruction is well established. Recent advances in understanding the mechanisms whereby calcium crystals contribute to cartilage damage are highlighted in this review. In vitro studies have shown that when calcium-containing crystals come in contact with cells they cause an influx in Ca 2+ concentration and activation of p42/44 mitogen-activated protein kinases. This is followed by induction of proto-oncogenes (c- fos, c- jun ) and induction of the nuclear transcription factors activator protein-1 and nuclear factor-kappaB, which in turn lead to crystal-induced modulation of normal gene expression. Some of the downstream effects known to date include increased mitogenesis, up-regulation of members of the matrix metalloproteinase family, down-regulation of tissue inhibitor of metalloproteinase-1 and -2 in fibroblasts, induction of neutrophil chemotactic chemokines such as interleukin-8, activation and degranulation of neutrophils, and inhibition of neutrophil apoptosis. Because no known drug prevents or treats the consequences of basic calcium phosphate crystal deposition, an improved understanding of the molecular mechanisms leading to crystal-induced joint degeneration is essential to the development of a rational approach to target the consequences of crystal deposition.