Maria Tsoli

Activation of Thermogenesis in Brown Adipose Tissue and Dysregulated Lipid Metabolism Associated with Cancer Cachexia in Mice

Cancer cachexia/anorexia is a complex syndrome that involves profound metabolic imbalances and is directly implicated as a cause of death in at least 20% to 30% of all cancers. Brown adipose tissue (BAT) plays a key role in thermogenesis and energy balance and potentially contributes to the physiologic perturbations associated with cachexia. In this study, we investigated the impact of cachexia-inducing colorectal tumor on BAT in mice. We found that brown adipocytes were smaller and exhibited profound delipidation in cachectic tumor-bearing mice. Diurnal expression profiling of key regulators of lipid accumulation and fatty acid β-oxidation and their corresponding target genes revealed dramatic molecular changes indicative of active BAT. Increased Ucp1, Pbe, and Cpt1α expression at specific points coincided with higher BAT temperatures during the dark cycle, suggestive of a temporal stimulation of thermogenesis in cachexia. These changes persisted when cachectic mice were acclimatized to 28°C confirming inappropriate stimulation of BAT despite thermoneutrality. Evidence of inflammatory signaling also was observed in the BAT as an energetically wasteful and maladaptive response to anorexia during the development of cachexia.

Cancer cachexia: malignant inflammation, tumorkines, and metabolic mayhem

Cachexia has a devastating impact on survival and quality of life for many cancer patients. A better understanding of the underlying mechanisms leading to the complex metabolic defects of cachexia, coupled with effective treatment options, will improve management of wasting in cancer patients. The growing appreciation that cancer cachexia results from the spillover effects of cytokine production by tumors on the integrated regulation of energy balance in different organs identifies potential therapeutic options. However, targeting such tumorkines requires a comprehensive understanding of their normal as well as pathophysiological functions, especially the crosstalk between inflammatory signaling and metabolic dysregulation. Recent advances in characterizing the surprising parallels between obesity and cancer cachexia provide new insights into these apparently divergent syndromes.

Depletion of White Adipose Tissue in Cancer Cachexia Syndrome Is Associated with Inflammatory Signaling and Disrupted Circadian Regulation

Involuntary weight loss in patients with cancer is the hallmark of cancer cachexia. The etiology of cachexia is multifactorial involving loss of skeletal muscle and adipose tissue associated with high systemic levels of acute phase proteins and inflammatory cytokines. While muscle wasting overtly impacts on cancer patient quality of life, loss of lipid depots represents a sustained energy imbalance. In this study fat depletion was examined in Colon-26 model of cancer cachexia, which is a widely used rodent model of this syndrome. We investigated diurnal expression of circadian rhythm regulators as well as key mediators of energy metabolism and cytokine signaling. Mice bearing the C26 tumour exhibited reduced adipose mass, elevated adipose tissue lipolysis and a 5-fold increase in plasma levels of free fatty acids. These changes were associated with activated IL-6 signaling in WAT through a 3-fold increase in phosphorylated STAT3 and high SOCS3 gene expression levels. In addition perturbations in circadian regulation of lipid metabolism were also observed. Lipid catabolism did not appear to be influenced by the classical PKA pathway activating the lipase HSL. ATGL protein levels were elevated 2-fold in cachectic mice while 4-fold increase phosphorylated ACC and a 2-fold decrease in phosphorylated 4EBP1 was observed indicating that lipid metabolism is modulated by the ATGL & AMPK/mTOR pathways. This study provides evidence for activation of cytokine signaling and concomitant alterations in circadian rhythm and regulators of lipid metabolism in WAT of cachectic animals.

The alternatively spliced murine pregnane X receptor isoform, mPXRΔ171–211 exhibits a repressive action

The orphan nuclear receptor pregnane X receptor regulates enzymes and transport proteins involved in the detoxification and clearance of numerous endobiotic and xenobiotic compounds, including pharmaceutical agents. Multiple alternatively spliced pregnane X receptor isoforms have been identified which are significantly expressed in humans and mice (up to 30% of the total pregnane X receptor transcript), however, little is known about their biological action. We explored functional differences between the major mouse pregnane X receptor isoforms mPXR(431) and mPXR(Delta171-211) that lacks 41 amino acids adjacent to the ligand-binding pocket. Transient transfection assays showed that mPXR(Delta171-211) reduced the basal transcription of cytochrome P450 3A4 and the drug transporter P-glycoprotein/Multi Drug Resistance Protein 1 and directly repressed the regulatory effects of mPXR(431) on these genes. Replacement of the mPXR(Delta171-211) DNA-binding domain with that of GAL4 showed mPXR(Delta171-211) retained its repressive role independent of binding to PXR responsive elements located within the cytochrome P450 3A4 and Multi Drug Resistance Protein 1 regulatory regions. Use of the histone deacetylase inhibitor, trichostatin A, demonstrated that the repressive function of mPXR(Delta171-211) acts independently of histone acetylation state. Protein interaction assays revealed mPXR(Delta171-211) and mPXR(431) differentially bind the obligatory heterodimer partner retinoid X receptor. Furthermore, mPXR(431) and mPXR(Delta171-211) proteins could heterodimerize. These studies demonstrate that the variant mouse PXR isoform, mPXR(Delta171-211), has a distinct repressive function from mPXR(431) in regulating genes encoding important drug metabolizing enzymes and transport proteins.

Abstract 1611: The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting

Background: Despite the success of chemotherapy in improving the overall survival rate of childhood cancer, a number of types of children9s cancers still have dismal outcomes. Included here are high risk neuroblastomas, Diffuse Intrinsic Pontine Gliomas (DIPG), and infant leukemias with MLL translocations. New treatments for these aggressive childhood cancers are urgently needed. Evidence is emerging of the importance of alterations in chromatin modifier genes in pediatric cancers. In this regard, CBL0137 is a carbazole-based anti-cancer agent with a unique mechanism of action. It is an indirect inhibitor of the chromatin remodeling complex FACT (Facilitates Chromatin Transcription). Inhibition of FACT by CBL0137 modulates the activity of several transcription factors involved in cancer: NF-kB and HSF1 are suppressed, while p53 is activated (Science Transl Med, 2011). We have examined FACT expression in neuroblastoma, DIPG and MLL leukemia, as well as the efficacy of CBL0137 in preclinical models of these diseases. Methods: Expression of the FACT subunits, SSRP1 and SPT16, was examined in neuroblastoma, DIPG and MLL leukemia cells using RT-PCR and Western analysis. The clinical significance of SSRP1 and SPT16 was also analysed using expression array data on 650 primary untreated neuroblastomas. Colony-forming assays were used to study the effect of CBL0137, either alone or combined with chemotherapeutic drugs. Cohorts of neuroblastoma, DIPG and MLL leukemia xenografted mice, as well as neuroblastoma-prone TH-MYCN mice, were treated with CBL0137, alone or combined with chemotherapeutic drugs. Results: High levels of SSRP1 and SPT16 expression were observed in all three types of child cancer. In addition, in neuroblastoma, the two FACT subunits were associated with MYCN amplification, and were strongly predictive of poor outcome (p Conclusions: Targeting FACT offers a highly promising novel therapeutic approach for aggressive childhood cancers. The results for CBL0137 are as good or better than any chemotherapy regimens we have tested in our preclinical models, and a Phase I COG trial of this nongenotoxic agent in refractory pediatric cancer patients is currently being planned. Citation Format: Michelle Haber, Jayne Murray, Laura Gamble, Ashleigh Carnegie-Clark, Hannah Webber, Michelle Ruhle, Michelle J. Henderson, Shiloh Middlemass, Daniel Carter, Maria Tsoli, Anahid Ehteda, Sandy Simon, Andre Oberthuer, Matthias Fischer, Katerina Gurova, Catherine Burkhart, Andrei Purmal, Richard B. Lock, David Ziegler, Glenn M. Marshall, Andrei V. Gudkov, Murray D. Norris. The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2015-1611

Abstract 3431: βIII-tubulin in glioblastoma: An emerging multifactorial survival factor role in chemotherapy response and tumor formation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastoma (GBM) is an aggressive primary brain tumour with a very poor prognosis. Current treatment is usually surgical resection, radiotherapy and chemotherapy, however, GBMs highly resistant nature renders treatment ineffective. sIII-tubulin is a neuronal specific microtubule protein that is aberrantly expressed in GBM and it has been implicated in a range of epithelial cancers with poor prognosis and high aggressiveness1. In gliomas, its high expression correlates with higher tumour grade2, however the functional relevance of this expression is unknown. Our aim was to investigate the functional and mechanistic role of sIII-tubulin in GBM drug sensitivity and tumorigenesis. Methods: To study the functional role of βIII-tubulin in GBM, siRNA gene knockdown was used in two GBM cell lines, U87 and U87vIII and in a primary GBM neurosphere culture. Non-targeting siRNA was used as a control. Knockdown levels were assessed by western blotting. Apoptosis was measured by AnnexinV staining. Drug sensitivity of cell lines was tested using drug-treated clonogenic assays. Senescence was assessed by using cytoplasmic staining for sa-β-galactosidase. Neurosphere formation was assessed by soft-agar growth assay. Results: Specific βIII-tubulin knockdown achieved a 94% and 52% reduction in sIII-tubulin levels in GBM cell lines and neurospheres respectively, compared to control siRNA. βIII-tubulin knockdown in U87vIII cells resulted in a significant increase in sensitivity to both the DNA-damaging agent temozolomide (TMZ) and tubulin binding agents epothilone B and paclitaxel compared to control (p<0.01). Increased sensitivity to TMZ following βIII-tubulin knockdown in U87 and U87vIII cells was associated with a significant increase in drug-induced apoptosis (p<0.01). TMZ is known to induce senescence3,4 and βIII-tubulin knockdown significantly increased TMZ-induced senescence in U87 (βIII-tub siRNA: 84.1±2.6%, p<0.05, compared to ctrl siRNA: 58.1±7.1%) and U87vIII (βIII-tub siRNA: 83.9±4.5%, p<0.01, compared to ctrl siRNA: 56.3±4.3%) cells. Further validation using a 3D-growth model of primary human GBM neurospheres, showed that neurosphere formation was significantly reduced (30.7%, p<0.01) upon βIII-tubulin knockdown compared to ctrl siRNA. Conclusions: Our data strongly suggests that aberrant expression of βIII-tubulin in glioblastoma may play an important role in intrinsic drug sensitivity to broad classes of chemotherapy drugs. Targeting βIII-tubulin increases temozolomide sensitivity by enhancing drug-induced apoptosis and senescence. Furthermore, we have identified a potential role for βIII-tubulin in GBM tumorigenesis. 1 Kavallaris. Nature Rev Cancer, 10:194-204, 2010 2 Katsetos et al. Arch Pathol Lab Med, 125:613-624, 2001 3 Mhaidat. Br J Cancer, 97:1225-1233, 2007 4 Gunther. Br J Cancer, 88:463-469, 2003 Citation Format: Gorjana Mitic, Maria Tsoli, David S. Ziegler, Maria Kavallaris. βIII-tubulin in glioblastoma: An emerging multifactorial survival factor role in chemotherapy response and tumor formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3431. doi:10.1158/1538-7445.AM2013-3431