The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis after exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered NH(2)-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

Androgen deprivation therapy for the treatment of prostate cancer: consider both benefits and risks

CONTEXT: Androgen deprivation therapy (ADT) is increasingly used for the treatment of prostate cancer (PCa), even in clinical settings in which there is no evidence-based proof of prolonged overall survival (OS). ADT, however, may be associated with numerous side effects, including an increased therapy-related cardiovascular mortality. OBJECTIVE: To discuss different clinical settings in which ADT is currently used and to critically weigh the benefits of ADT against its possible side effects. EVIDENCE ACQUISITION: A MEDLINE search was conducted to identify original articles and review articles addressing the efficacy and side effects of ADT for the treatment of PCa. Keywords consisted of prostate cancer, hormonal therapy, adverse effects, radical prostatectomy, and radiotherapy. The articles with the highest level of evidence for the various examined end points were identified with the consensus of all authors and were reviewed. EVIDENCE SYNTHESIS: Even short-term use of ADT may lead to numerous side effects, such as osteoporosis, obesity, sarcopenia, lipid alterations, insulin resistance, and increased risk for diabetes and cardiovascular morbidity. Despite these side effects, ADT is commonly used in various clinical settings in which a clear effect on improved OS has not been shown. CONCLUSIONS: ADT is associated with an increased risk of multiple side effects that may reduce quality of life and/or OS. Consequently, these issues should be discussed in detail with patients and their families before initiation of ADT. ADT should be used with knowledge of its potential long-term side effects and with possible lifestyle interventions, especially in settings with the highest risk-benefit ratio, to alleviate comorbidities.

miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer

MicroRNAs (miRNA) are negative regulators of gene expression at the posttranscriptional level, which are involved in tumorigenesis. Two miRNAs, miR-15a and miR-16, which are located at chromosome 13q14, have been implicated in cell cycle control and apoptosis, but little information is available about their role in solid tumors. To address this question, we established a protocol to quantify miRNAs from laser capture microdissected tissues. Here, we show that miR-15a/miR-16 are frequently deleted or down-regulated in squamous cell carcinomas and adenocarcinomas of the lung. In these tumors, expression of miR-15a/miR-16 inversely correlates with the expression of cyclin D1. In non-small cell lung cancer (NSCLC) cell lines, cyclins D1, D2, and E1 are directly regulated by physiologic concentrations of miR-15a/miR-16. Consistent with these results, overexpression of these miRNAs induces cell cycle arrest in G(1)-G(0). Interestingly, H2009 cells lacking Rb are resistant to miR-15a/miR-16-induced cell cycle arrest, whereas reintroduction of functional Rb resensitizes these cells to miRNA activity. In contrast, down-regulation of Rb in A549 cells by RNA interference confers resistance to these miRNAs. Thus, cell cycle arrest induced by these miRNAs depends on the expression of Rb, confirming that G(1) cyclins are major targets of miR-15a/miR-16 in NSCLC. Our results indicate that miR-15a/miR-16 are implicated in cell cycle control and likely contribute to the tumorigenesis of NSCLC.

Autophagy in cancer and chemotherapy

Cancer cells often exhibit mutations in critical molecules of the apoptotic machinery, resulting in resistance to common anticancer therapies. In the absence of apoptosis, autophagic cell death can be an alternative form of cell death by excessive self-digestion. Therefore, autophagic cell death can be considered as a backup cell death mechanism when apoptotic cell death mechanisms fail. However, many tumors also exhibit deficiencies in autophagy that may result in both genomic instability and further anticancer drug resistance. This chapter summarizes our current understanding regarding the regulation of autophagy in tumors and discusses potential new anticancer drug treatment strategies.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Bacteria causing bacteremia in pediatric cancer patients presenting with febrile neutropenia – species distribution and susceptibility patterns

PURPOSE Infections are a major cause of morbidity and mortality in pediatric cancer patients. The aim of this study was to establish the microbiological spectrum and the susceptibility patterns of bacteremia-causing bacteria in pediatric cancer patients with febrile neutropenia in relation to the use of prophylactic and empirical antibiotics. METHODS We analyzed positive blood cultures of pediatric cancer patients presenting with febrile neutropenia between 2004 and 2011 in Groningen and Amsterdam (the Netherlands) and in Bern (Switzerland), using different antibiotic prophylactic and empirical regimens. RESULTS A total of 156 patients with 202 bacteremias, due to 248 bacteria species, were enrolled. The majority (73%) of bacteremias were caused by Gram-positive bacteria. Gram-negative bacteria, especially Pseudomonas aeruginosa, were observed significantly more often in Bern, where no fluoroquinolone prophylaxis was used. Ciprofloxacin-resistant bacteria were cultured more often from patients who did receive ciprofloxacin prophylaxis, compared to the patients who did not (57 versus 11%, p = 0.044). CONCLUSIONS Gram-positive bacteria predominated in this study. We showed that the use of prophylactic antibiotics in pediatric cancer patients was associated with increased resistance rates, which needs further study. The strategy for empiric antimicrobial therapy for febrile neutropenia should be adapted to local antibiotic resistance patterns.

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

Biological, diagnostic and therapeutic relevance of the MET receptor signaling in head and neck cancer.

Head and neck cancer constitutes the 6th most common malignancy worldwide and affects the crucial anatomical structures and physiological functions of the upper aerodigestive tract. Classical therapeutic strategies such as surgery and radiotherapy carry substantial toxicity and functional impairment. Moreover, the loco-regional control rates as well as overall survival still need to be improved in subgroups of patients. The scatter-factor/hepatocyte growth factor receptor tyrosine kinase MET is an established effector in the promotion, maintenance and progression of malignant transformation in a wide range of human malignancies, and has been gaining considerable interest in head and neck cancer over the last 15 years. Aberrant MET activation due to overexpression, mutations, tumor-stroma paracrine loops, and cooperative/redundant signaling has been shown to play prominent roles in epithelial-to-mesenchymal transition, angiogenesis, and responses to anti-cancer therapeutic modalities. Accumulating preclinical and translational evidence highly supports the increasing interest of MET as a biomarker for lymph node and distant metastases, as well as a potential marker of stratification for responses to ionizing radiation. The relevance of MET as a therapeutic molecular target in head and neck cancer described in preclinical studies remains largely under-evaluated in clinical trials, and therefore inconclusive. Also in the context of anti-cancer targeted therapy, a large body of preclinical data suggests a central role for MET in treatment resistance towards multiple therapeutic modalities in malignancies of the head and neck region. These findings, as well as the potential use of combination therapies including MET inhibitors in these tumors, need to be further explored.

Synthesis and cellular characterization of novel isoxazolo- and thiazolohydrazinylidene-chroman-2,4-diones on cancer and non-cancer cell growth and death

Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the synthesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by derivatization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS. These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231, PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48 h and 72 h of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC50 values) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e were found to be the most promising and effective ones having EC50 values that were several fold reduced when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell viability was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the survival protein kinase Akt. In summary, this study has identified three novel coumarin derivatives that in comparison to 4-hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and therefore may represent interesting novel drug candidates

TWIST1 and TWIST2 promoter methylation and protein expression in tumor stroma influence the epithelial-mesenchymal transition-like tumor budding phenotype in colorectal cancer.

Tumor budding in colorectal cancer is likened to an epithelial-mesenchymal transition (EMT) characterized predominantly by loss of E-cadherin and up-regulation of E-cadherin repressors like TWIST1 and TWIST2. Here we investigate a possible epigenetic link between TWIST proteins and the tumor budding phenotype. TWIST1 and TWIST2 promoter methylation and protein expression were investigated in six cell lines and further correlated with tumor budding in patient cohort 1 (n = 185). Patient cohort 2 (n = 112) was used to assess prognostic effects. Laser capture microdissection (LCM) of tumor epithelium and stroma from low- and high-grade budding cancers was performed. In colorectal cancers, TWIST1 and TWIST2 expression was essentially restricted to stromal cells. LCM results of a high-grade budding case show positive TWIST1 and TWIST2 stroma and no methylation, while the low-grade budding case was characterized by negative stroma and strong hypermethylation. TWIST1 stromal cell staining was associated with adverse features like more advanced pT (p = 0.0044), lymph node metastasis (p = 0.0301), lymphatic vessel invasion (p = 0.0373), perineural invasion (p = 0.0109) and worse overall survival time (p = 0.0226). Stromal cells may influence tumor budding in colorectal cancers through expression of TWIST1. Hypermethylation of the tumor stroma may represent an alternative mechanism for regulation of TWIST1.

Tyrosine kinase receptor B (TrkB) expression in colorectal cancers highlights anoikis resistance as a survival mechanism of tumour budding cells.

AIMS Tumour buds in colorectal cancer represent an aggressive subgroup of non-proliferating and non-apoptotic tumour cells. We hypothesize that the survival of tumour buds is dependent upon anoikis resistance. The role of tyrosine kinase receptor B (TrkB), a promoter of epithelial-mesenchymal transition and anoikis resistance, in facilitating budding was investigated. METHODS AND RESULTS Tyrosine kinase receptor B immunohistochemistry was performed on a multiple-punch tissue microarray of 211 colorectal cancer resections. Membranous/cytoplasmic and nuclear expression was evaluated in tumour and buds. Tumour budding was assessed on corresponding whole tissue slides. Relationship to Ki-67 and caspase-3 was investigated. Analysis of Kirsten Ras (KRAS), proto-oncogene B-RAF (BRAF) and cytosine-phosphate-guanosine island methylator phenotype (CIMP) was performed. Membranous/cytoplasmic and nuclear TrkB were strongly, inversely correlated (P < 0.0001; r = -0.41). Membranous/cytoplasmic TrkB was overexpressed in buds compared to the main tumour body (P < 0.0001), associated with larger tumours (P = 0.0236), high-grade budding (P = 0.0011) and KRAS mutation (P = 0.0008). Nuclear TrkB was absent in buds (P <0.0001) and in high-grade budding cancers (P =0.0073). Among patients with membranous/cytoplasmic TrkB-positive buds, high tumour membranous/cytoplasmic TrkB expression was a significant, independent adverse prognostic factor [P = 0.033; 1.79, 95% confidence interval (CI) 1.05-3.05]. Inverse correlations between membranous/cytoplasmic TrkB and Ki-67 (r = -0.41; P < 0.0001) and caspase-3 (r =-0.19; P < 0.05) were observed. CONCLUSIONS Membranous/cytoplasmic TrkB may promote an epithelial-mesenchymal transition (EMT)-like phenotype with high-grade budding and maintain viability of buds themselves.

Dsh homolog DVL3 mediates resistance to IGFIR inhibition by regulating IGF-RAS signaling

Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition.

BRCA2-Deficient Sarcomatoid Mammary Tumors Exhibit Multidrug Resistance.

Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this. Cancer Res; 75(4); 732-41. ©2014 AACR.