Oral Lichen Planus and Mutated TP53-A Road to Cancer?

The malignant potential of oral lichen planus (OLP) has been discussed and disputed for decades. The lesions are often characterized by strong expression of the TP53 protein in the basal layer of the mucosa. In 2002, we reported the presence of TP53 mutations in nine out of 27 OLP lesions tested. At follow-up in 2009, one case of oral squamous cell cancer (OSCC) had occurred in a different site six years later. In contrast, in another case, TP53 mutation persisted for years without malignant transformation. In a longitudinal study of eight selected patients with OSCC or different pre-malignant lesions, it was concluded that TP53 mutations could occur early or late in the development of OSCC. A follow-up in the present, almost 20 years later, revealed that one further case of OSCC had occurred in a TP53-mutated case of OLP, 21 years after the first sample was taken, again in a different site. With this second case, this small study now points towards a risk of developing OSCC in TP53-mutated OLP lesions. A review of recent literature indicates a growing consensus that OLP should be regarded as a potentially pre-malignant lesion. Several protein markers have been studied, but none proved useful for prediction of malignant progression. The great majority of published studies are retrospective, and it has been suggested that multi-centre prospective studies will be needed to reach a definitive answer on the malignant potential of OLP, and particularly, to identify contributing factors. Screening for TP53 mutations could help to identify the subgroup of OLP patients that is truly at risk of developing oral cancer.

Cytotoxic effects of halogenated tin phosphinoyldithioformate complexes against several cancer cell lines.

Organotin complexes are studied as promising alternatives to the anticancer drug cisplatin. We report two monoorganotin(IV) complexes based on a dibenzyl phosphinoyldithioformate (H-DBPTF) ligand, containing either bromide (Sn-DBPTF-1) or chloride (Sn-DBPTF-2) anions. The complexes were characterized by standard analytical techniques and the structural details of these complexes were elucidated by single crystal X-ray diffraction. Sn-DBPTF-1 was cytotoxic at IC50 <10 µg mL-1 against cancer cell lines A549 (lung cancer), Aspc-1 (pancreatic cancer), OVCAR-3 (ovarian cancer), T-47D (breast cancer) and HCT116 (colon cancer), and breast epithelial stem cell line D492. The non-tumorigenic breast epithelial cell line MCF-10 was less sensitive at IC50 = 22 µg mL-1. Sn-DBPTF-2 had limited cytotoxic effect at IC50 13-37 µg mL-1. Sn-DBPTF-1 induced apoptosis and double-strand DNA breaks. Cell cycle arrest in G2 occurred in HCT116 and accumulation in G1 in Aspc-1. The results indicate that the basic effect of Sn-DBPTF-1 is to induce DNA damage, leading to apoptosis and cell cycle arrest depending on the cell line.

The Anti-Proliferative Lichen-Compound Protolichesterinic Acid Inhibits Oxidative Phosphorylation and Is Processed via the Mercapturic Pathway in Cancer Cells.

The lichen compound protolichesterinic acid (PA) has an anti-proliferative effect against several cancer cell lines of different origin. This effect cannot be explained by the known inhibitory activity of PA against 5- and 12-lipoxygenases. The aim was therefore to search for mechanisms for the anti-proliferative activity of PA. Two cancer cell lines of different origin, both sensitive to anti-proliferative effects of PA, were selected for this study, T-47D from breast cancer and AsPC-1 from pancreatic cancer. Morphological changes were assessed by transmission electron microscopy, HPLC coupled with TOF spectrometry was used for metabolomics, mitochondrial function was measured using the Agilent Seahorse XFp Real-time ATP assay and glucose/lactate levels by radiometry. Levels of glutathione, NADP/NADPH and reactive oxygen species [ROS] were measured by luminescence. Following exposure to PA both cell lines showed structural changes in mitochondria that were in line with a measured reduction in oxidative phosphorylation and increased glycolysis. These changes were more marked in T-47D, which had poorer mitochondrial function at baseline. PA was processed and expelled from the cells via the mercapturic pathway, which consumes glutathione. Nevertheless, glutathione levels were increased after 24 hours of exposure to PA, implying enhanced synthesis. Redox balance was not much affected and ROS levels were not increased. We conclude that PA is metabolically processed and expelled from cells, leading indirectly to increased glutathione levels with minimal effects on redox balance. The most marked effect was on mitochondrial structure and metabolic function implying that effects of PA may depend on mitochondrial fitness.

Use of proton pump inhibitors and mortality among Icelandic patients with prostate cancer.

Proton pump inhibitors (PPIs) are commonly used drugs among cancer patients. Due to conflicting reports on their safety, we aimed to determine whether PPI use is associated with mortality among prostate cancer patients. In this population-based cohort study, we identified incident diagnoses of prostate cancer between 2007 and 2012 (n = 1058). Follow-up was from 12 months after diagnosis until death, emigration or end the of study. Post-diagnosis use was defined as ≥2 filled prescriptions following diagnosis. We used time-dependent Cox proportional hazard regression models to compute hazard ratios (HRs) and 95% confidence intervals (CIs) for prostate cancer-specific and all-cause mortality associated with post-diagnosis use of PPIs. We identified 347 (32.8%) post-diagnosis PPI users and 711 (67.2%) non-users after diagnosis. Of the 347 patients using PPIs after diagnosis, 59 (17.0%) died due to any cause and 22 (6.3%) due to prostate cancer, compared with 144 (20.3%) and 76 (10.7%) among non-users after diagnosis, respectively. Post-diagnosis PPI use was not associated with prostate cancer-specific mortality (HR 0.88; 95% CI: 0.52-1.48) or all-cause mortality (HR 1.02; 95% CI: 0.73-1.43). Contrary to a previous report, this study did not find evidence of an association between post-diagnosis PPI use and mortality among prostate cancer patients.

MITF has a central role in regulating starvation-induced autophagy in melanoma.

The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis. The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma. Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells. The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding. In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3. Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux. Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma. Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.

Proton pump inhibitor use and risk of breast cancer, prostate cancer, and malignant melanoma: An Icelandic population-based case-control study.

PURPOSE: Increased expression of Vacuolar-type H+ ATPases (V-ATPases), in the plasma membrane of cancer cells has been suggested to contribute to the development of aggressive cancer phenotypes by promoting acidic tumor microenvironments. Accumulating data suggest that proton pump inhibitors (PPIs) may elicit a chemopreventive effect via V-ATPase inhibition in some cancers, but evidence is still limited. Therefore, we aimed to explore a potential preventive role of PPIs in this study. METHODS: In this population-based case-control study, we identified incident cases of breast cancer (n = 1739), prostate cancer (n = 1897), and malignant melanoma (n = 385) in Iceland between 2005 and 2014 from the Icelandic Cancer Registry. We assessed varying levels of PPI use through record linkages to the Icelandic Medicines Registry. For each case, we selected up to 10 age-matched, sex-matched, and calendar-matched population controls using risk-set sampling. Using conditional logistic regression, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) controlling for NSAID use. RESULTS: Adjusted ORs associated with ever use of PPIs were 1.03 (95% CI: 0.92-1.16) for breast cancer, 1.12 (95% CI: 1.00-1.25) for prostate cancer, and 0.84 (95% CI: 0.69-1.12) for malignant melanoma. Analyses of high use of PPIs (≥1000 DDDs) yielded ORs of 0.97 (95% CI: 0.78-1.19), 1.20 (0.99-1.47), and 0.59 (0.40-1.13) for breast cancer, prostate cancer, and malignant melanoma, respectively. Analyses of cumulative exposure to PPIs did not support a dose-response relationship for any of the three cancer types. CONCLUSIONS: Our findings do not support a chemopreventive effect of PPI use on breast cancer, prostate cancer, or malignant melanoma.

Altered plasmalogen content and fatty acid saturation following epithelial to mesenchymal transition in breast epithelial cell lines.

Epithelial to mesenchymal transition (EMT) is a developmental event characterized by phenotypic switching from a polarized epithelial phenotype to an unpolarized mesenchymal phenotype. Changes to plasma membrane function accompany EMT yet the differences in lipid composition of cells that have undergone EMT are relatively unexplored. To address this the lipidome of two cell models of EMT in breast epithelial tissue, D492 and HMLE, were analyzed by untargeted LC-MS. Detected masses were identified and their abundance was compared through multivariate statistical analysis. Considerable concordance was observed in eight lipid components between epithelial and mesenchymal cells in both cell models. Specifically, an increase in phosphatidylcholine and triacylglycerol were found to accompany EMT while phosphatidylcholine- and phosphatidylethanolamine plasmalogens, as well as diacylglycerols decreased. The most abundant fatty acid lengths were C16 and C18 but mesenchymal cells had on average shorter and more unsaturated fatty acids. The results are consistent with enhanced cell mobility post EMT and reflect a consequence of oxidative stress pre- and post EMT in breast epithelial tissue.

Proton-pump inhibitors among adults: a nationwide drug-utilization study.

BACKGROUND: The use of proton-pump inhibitors (PPIs) has grown worldwide, and there are concerns about increased unsubstantiated long-term use. The aim of the study was to describe the real-world use of PPIs over the past decade in an entire national population. METHODS: This was a nationwide population-based drug-utilization study. Patterns of outpatient PPI use among adults in Iceland between 2003 and 2015 were investigated, including annual incidence and prevalence, duration of use, and dose of tablet used (lower versus higher), as well as the proportion of PPI use attributable to gastroprotection. RESULTS: We observed 1,372,790 prescription fills over the entire study period, of which 95% were for higher-dose PPIs. Annual incidence remained stable across time (3.3-4.1 per 100 persons per year), while the annual prevalence increased from 8.5 per 100 persons to 15.5 per 100 persons. Prevalence increased with patient age and was higher among women than men. Duration of treatment increased with patients' age (36% of users over 80 years remained on treatment after 1 year compared with 13% of users aged 19-39 years), and was longer among those initiating on a higher dose compared with a lower dose. The proportion of PPI users concurrently using nonsteroidal anti-inflammatory drugs decreased over the study period, while the proportion concurrently using acetylsalicylic acid, oral anticoagulants, or platelet inhibitors increased. CONCLUSIONS: In this nationwide study, a considerable increase in overall outpatient use of PPIs over a 13-year period was observed, particularly among older adults. Patients were increasingly treated for longer durations than recommended by clinical guidelines and mainly with higher doses.

Metabolic Profiling as a Screening Tool for Cytotoxic Compounds: Identification of 3-Alkyl Pyridine Alkaloids from Sponges Collected at a Shallow Water Hydrothermal Vent Site North of Iceland.

Twenty-eight sponge specimens were collected at a shallow water hydrothermal vent site north of Iceland. Extracts were prepared and tested in vitro for cytotoxic activity, and eight of them were shown to be cytotoxic. A mass spectrometry (MS)-based metabolomics approach was used to determine the chemical composition of the extracts. This analysis highlighted clear differences in the metabolomes of three sponge specimens, and all of them were identified as Haliclona (Rhizoniera) rosea (Bowerbank, 1866). Therefore, these specimens were selected for further investigation. Haliclona rosea metabolomes contained a class of potential key compounds, the 3-alkyl pyridine alkaloids (3-APA) responsible for the cytotoxic activity of the fractions. Several 3-APA compounds were tentatively identified including haliclamines, cyclostellettamines, viscosalines and viscosamines. Among these compounds, cyclostellettamine P was tentatively identified for the first time by using ion mobility MS in time-aligned parallel (TAP) fragmentation mode. In this work, we show the potential of applying metabolomics strategies and in particular the utility of coupling ion mobility with MS for the molecular characterization of sponge specimens.

Effects of anti-proliferative lichen metabolite, protolichesterinic acid on fatty acid synthase, cell signalling and drug response in breast cancer cells.

BACKGROUND: The lichen compound (+)-protolichesterinic acid (+)-PA, isolated from Iceland moss, has anti-proliferative effects on several cancer cell lines. The chemical structure of (+)-PA is similar to a known fatty acid synthase (FASN) inhibitor C75. AIMS: To test whether the anti-proliferative activity of (+)-PA is associated with effects on FASN and HER2 (human epidermal growth factor receptor 2) and major signalling pathways. Synergism between (+)-PA and lapatinib, a HER2 active drug, was also evaluated. MATERIALS AND METHODS: Pure compound was isolated by preparative high-performance liquid chromatography (HPLC) and purity of (+)-PA analyzed by analytical HPLC. Cell viability was assessed using Crystal violet staining. FASN and HER2 expression was estimated by immunofluorescence. The Meso Scale Discovery (MSD)(®) assay was used to measure activation of ERK1/2 and AKT. Synergism was estimated by the CalcuSyn software. RESULTS: Treatment with (+)-PA increased FASN expression in SK-BR-3 cells, which overexpress FASN and HER2, implying a compensatory response to inhibition of FASN activity. HER2 expression was decreased suggesting secondary downregulation. ERK1/2 and AKT signalling pathways were inhibited, probably due to reduced levels of HER2. No effects were observed in T-47D cells. Synergism between (+)-PA and lapatinib was observed in the SK-BR-3 cells. CONCLUSION: Results suggest that the primary effect of (+)-PA is inhibition of FASN activity. Synergistic effects with lapatinib were seen only in SK-BR-3 cells, and not T-47D cells, further supporting the notion that (+)-PA acts by inhibiting FASN with secondary effects on HER2 expression and signalling. (+)-PA could therefore be a suitable agent for further testing, alone or in combination treatment against HER2-overexpressing breast cancer.

Inherited polymorphisms in hyaluronan synthase 1 predict risk of systemic B-cell malignancies but not of breast cancer.

Genetic variations in the hyaluronan synthase 1 gene (HAS1) influence HAS1 aberrant splicing. HAS1 is aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM), but not in their counterparts from healthy donors. The presence of aberrant HAS1 splice variants predicts for poor survival in multiple myeloma (MM). We evaluated the influence of inherited HAS1 single nucleotide polymorphisms (SNP) on the risk of having a systemic B cell malignancy in 1414 individuals compromising 832 patients and 582 healthy controls, including familial analysis of an Icelandic kindred. We sequenced HAS1 gene segments from 181 patients with MM, 98 with monoclonal gammopathy of undetermined significance (MGUS), 72 with Waldenstrom macroglobulinemia (WM), 169 with chronic lymphocytic leukemia (CLL), as well as 34 members of a monoclonal gammopathy-prone Icelandic family, 212 age-matched healthy donors and a case-control cohort of 295 breast cancer patients with 353 healthy controls. Three linked single nucleotide polymorphisms (SNP) in HAS1 intron3 are significantly associated with B-cell malignancies (range p = 0.007 to p = 10(-5)), but not MGUS or breast cancer, and predict risk in a 34 member Icelandic family (p = 0.005, Odds Ratio = 5.8 (OR)), a relatively homogeneous cohort. In contrast, exon3 SNPs were not significantly different among the study groups. Pooled analyses showed a strong association between the linked HAS1 intron3 SNPs and B-cell malignancies (OR = 1.78), but not for sporadic MGUS or for breast cancer (OR<1.0). The minor allele genotypes of HAS1 SNPs are significantly more frequent in MM, WM, CLL and in affected members of a monoclonal gammopathy-prone family than they are in breast cancer, sporadic MGUS or healthy donors. These inherited changes may increase the risk for systemic B-cell malignancies but not for solid tumors.

Familial predisposition to monoclonal gammopathies: deviations in B-cell biology.

Monoclonal gammopathies are associated with advancing age but a familial predisposition has been recognized for several decades. A functional phenotype, characterized by increased immunoglobulin (Ig) production after mitogen stimulation has been identified in healthy members of 4 families showing a predisposition toward IgM and IgG/IgA disorders. B cells from these hyperresponders do not show increased rates of Ig gene translocations and no aberrations were detected in an in vitro model of the germinal center reaction. Array-based comparative genome hybridization revealed deletions of Ig genes in peripheral blood B cells, as expected. In addition, random changes were detected throughout the genome, presumably reflecting off-target activation-induced cytidine deaminase (AID) activity. These random changes were significantly less prevalent in B cells from hyperresponders, indicating less exposure to the germinal center environment during maturation.

Proton-shuttling lichen compound usnic acid affects mitochondrial and lysosomal function in cancer cells.

The lichen compound usnic acid (UA) is a lipophilic weak acid that acts as a proton shuttle and causes loss of mitochondrial inner membrane potential. In the current study we show that UA treatment induced the formation of autophagosomes in human cancer cells, but had minimal effects on normal human fibroblasts. However, autophagic flux was incomplete, degradation of autophagosomal content did not occur and acidification was defective. UA-treated cells showed reduced ATP levels and activation of AMP kinase as well as signs of cellular stress. UA is thus likely to trigger autophagosome formation both by energy depletion and stress conditions. Our findings indicate that the H(+)-shuttling effect of UA operates not only in mitochondria as previously shown, but also in lysosomes, and have implications for therapeutic manipulation of autophagy and pH-determined drug distribution.

Synergistic cytotoxic effect of the microtubule inhibitor marchantin A from Marchantia polymorpha and the Aurora kinase inhibitor MLN8237 on breast cancer cells in vitro.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC50 = 5.5 µM), MCF7 (IC50 = 11.5 µM), and T47D (IC50 = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator.

Tetraploidy in BRCA2 breast tumours.

Tetraploidy and aneuploidy can be caused by cell division errors and are frequently observed in many human carcinomas. We have recently reported delayed cytokinesis in primary human fibroblasts from BRCA2 mutation carriers, implying a function for the BRCA2 tumour suppressor in completion of cell division. Here, we address ploidy aberrations in breast tumours derived from BRCA2 germline mutation carriers. Ploidy aberrations were evaluated from flow cytometry histograms on selected breast tumour samples (n=236), previously screened for local BRCA mutations. The ploidy between BRCA2-mutated (n=71) and matched sporadic (n=165) cancers was compared. Differences in ploidy distribution were examined with respect to molecular tumour subtypes, previously defined by immunohistochemistry on tissue microarray sections. Tetraploidy was significantly 3 times more common in BRCA2 breast cancers than sporadic. However, no differences were found in the overall ploidy distribution between BRCA2-mutation carriers and non-carriers. In BRCA2 cancers, tetraploidy was associated with luminal characteristics. The increased frequency of tetraploidy in BRCA2 associated cancers may be linked to cell division errors, particularly cytokinesis. Additionally, tetraploidy emerges predominantly in BRCA2 breast cancers displaying luminal rather than triple-negative phenotypes.

Cytogenetic polyclonality of breast carcinomas: association with clinico-pathological characteristics and outcome.

Routinely used prognostic factors fail to predict clinical outcome in a significant proportion of breast cancer patients, implying that they can not detect some important biological characteristics. Chromosomal changes have been described in breast carcinomas for many years but their significance is not clear. We compared chromosomal changes with clinico-pathological characteristics and clinical outcome in 203 breast cancer patients with a follow-up of 9-18 years. Combining data from classical cytogenetics and flow cytometry revealed chromosomal abnormalities in 142 cases (70%). Of these, 51 (35.9%) contained two or more cytogenetically abnormal clones. Polyclonality was significantly associated with poor breast-cancer-specific survival (P = 0.03) within 5 years, independent of tumor size, lymph node metastases, and hormone receptors. Specific changes were similar to those previously described, but a new finding was a significant association between del 3p12p21 and poor survival. Polyclonality was significantly associated with TP53-mutations but not with a germline BRCA2 mutation. Less than one third of the polyclonal samples were identified by flow cytometry alone. Cytogenetic changes were detected in 17 out of 114 samples from non-tumorous tissue (15%), two of them identical with a clone in the corresponding tumor. Several samples contained clearly unrelated clones within the tumor and outside, implying either multifocal origin or early divergence. In conclusion, the common deletion on Chromosome 3p12p21 was associated with poor clinical outcome. Chromosomal polyclonality is common in breast carcinomas and predicts poor survival. Polyclonality was poorly detected by one-sample flow cytometry. Multiple sampling might improve the detection rate.

Familial paraproteinemia: hyper-responsive B-cells as endophenotype.

The prevalence of paraproteinemias or monoclonal gammopathies increases with age. No other major risk factors have been recognized, but significant associations have been reported with chronic antigen exposure, agricultural environment, and family history. In around 130 families reported worldwide, IgG or IgA monoclonal gammopathy of undetermined significance (MGUS) occurs with multiple myeloma (MM) whereas Waldenström's macroglobulinemia (WM) is linked to IgM MGUS. Of the 8 multi-case families described here, 5 are remarkable for including both IgG/IgA and IgM type disorders. In the remaining 3 families IgG/IgA MGUS and MM occurred with Hodgkin disease and T-cell malignancies. These different patterns of familial paraproteinemia indicate different genetic backgrounds. A previously described functional phenotype of hyper-responsive B lymphocytes fulfils criteria for being an endophenotype and may be related to raised serum IgM. Identifying an endophenotype is important to ensure correct classification of affected family members and thus enhance the power of genetic studies.