Ovarian cancer (OvCa) exhibits a specific predilection for metastasis to the omentum. Our earlier studies highlighted the tumour-suppressor effect of secreted protein acidic and rich in cysteine ...(SPARC) in OvCa through multi-faceted roles inhibiting cancer cell interactions within the peritoneal milieu. The goal of this study is to investigate the role of SPARC in OvCa interactions with omental adipocytes and its role in OvCa colonization in the omentum. We employed multi-pronged approach using primary omental adipocytes from Sparc knockout mice, genetically engineered human omental adipocytes in 3D co-cultures with OvCa cells, as well as treatment with recombinant SPARC protein. We show that SPARC suppresses multistep cascade in OvCa omental metastasis. SPARC inhibited in vivo and adipocyte-induced homing, proliferation, and invasion of OvCa cells. SPARC suppressed metabolic programming of both adipocytes and OvCa cells and exerted an inhibitory effect of adipocyte differentiation and their phenotypic switch to cancer-associated phenotype. Mechanistic studies revealed that this effect is mediated through inhibition of cEBPβ-NFkB-AP-1 transcription machinery. These findings define a novel and functionally important role of SPARC in OvCa and not only bridge the knowledge gap but highlight the need to consider SPARC protein expression in therapeutic development.
Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF‐1 in this context, the role of additional ...mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant‐negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.
The tropism of ovarian cancer (OvCa) to the peritoneal cavity is implicated in widespread dissemination, suboptimal surgery, and poor prognosis. This tropism is influenced by stromal factors that are ...not only critical for the oncogenic and metastatic cascades, but also in the modulation of cancer cell metabolic plasticity to fulfill their high energy demands. In this respect, we investigated the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in metabolic plasticity of OvCa. We used a syngeneic model of OvCa in
-deficient and proficient mice to gain comprehensive insight into the paracrine effect of stromal-SPARC in metabolic programming of OvCa in the peritoneal milieu. Metabolomic and transcriptomic profiling of micro-dissected syngeneic peritoneal tumors revealed that the absence of stromal-
led to significant upregulation of the enzymes involved in glycolysis, TCA cycle, and mitochondrial electron transport chain (ETC), and their metabolic intermediates. Absence of stromal-
increased reactive oxygen species and perturbed redox homeostasis. Recombinant SPARC exerted a dose-dependent inhibitory effect on glycolysis, mitochondrial respiration, ATP production and ROS generation. Comparative analysis with human tumors revealed that SPARC-regulated ETC-signature inversely correlated with SPARC transcripts. Targeting mitochondrial ETC by phenformin treatment of tumor-bearing
-deficient and proficient mice mitigated the effect of SPARC-deficiency and significantly reduced tumor burden, ROS, and oxidative tissue damage in syngeneic tumors. In summary, our findings provide novel insights into the role of SPARC in regulating metabolic plasticity and bioenergetics in OvCa, and shines light on its potential therapeutic efficacy.
1,25-dihydroxyvitamin D3, (1,25(OH)2D3; calcitriol), the hormonal form of vitamin D, exerts growth-inhibitory, pro-apoptotic and anti-metastatic effects on tumor cells in vitro and in vivo but its ...clinical use is limited by its calcemic effects. Previous studies have shown that the antiproliferative effects of the less calcemic calcitriol analog 19-nor-1,25-(OH)2D2 (paricalcitol) on prostate tumor cell lines are indistinguishable from those of 1,25(OH)2D3. We therefore investigated the anti-proliferative effects of paricalcitol on the growth of pancreatic tumor cell lines in vitro and in vivo. Both 1,25(OH)2D3 and paricalcitol inhibited the growth of BxPC-3, Hs700T and AsPC-1 lines in a dose-dependent manner. This antiproliferative activity correlated with upregulation of the cell-cycle inhibitors p21 (Waf1/CIP1) and p27(Kip1). A fourth pancreatic cell line, Hs766T was unresponsive to both paricalcitol and calcitriol. Hs766T cells also failed to upregulate p21/Waf-1/Cip1 or p27/KiP in response to treatments with these agents. Paricalcitol, given three times per week inhibited the growth of AsPC-1 pancreatic tumor cell xenografts in nude mice at a dose that did not cause hypercalcaemia. Tumor inhibition was accompanied by in vivo upregulation of p21 and p27 expression. Given the few therapeutic options for patients with pancreatic cancer, further exploration of paricalcitol, an F.D.A. approved medication, is warranted.
The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break repair (DSBR) ...proteins. Most of the known activities of E4orf6 during adenoviral infection require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6, stably expressed in RKO human colorectal carcinoma cells or transiently expressed by adenoviral vector in U251 human glioblastoma cells, inhibits DSBR and induces significant radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P) failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (γ-H2AX) levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid (pSP189) in 293 cells but did not increase the mutation frequency measured in the reporter plasmid. The kinase activity of DNA-PKcs (the DNA-dependent protein kinase catalytic subunit) toward heterologous substrates was not affected by expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr-2609 following ionizing radiation was prolonged in the presence of E4orf6 when compared with control-infected cells. Our results demonstrate for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian cells in the absence of E1B-55K or other adenoviral genes and suggest that viral-mediated delivery of E4orf6, combined with localized external beam radiation, could be a useful approach for the treatment of radioresistant solid tumors such as glioblastomas.
Abstract
We have reported a tumor suppressor effect of SPARC in ovarian cancer as both host and tumor SPARC are implicated in anti-proliferative, anti-adhesive effects as well as normalization of the ...peritoneal tumor microenvironment. Herein, we extend our studies using a syngeneic model of peritoneal ovarian carcinomatosis to gain comprehensive insight on the effect of SPARC on metabolic programming of the ovarian cancer ecosystem. We performed comprehensive metabolomic and transcriptomic profiling of micro-dissected ID8 omental tumors that developed after intra-peritoneal injection in SP-/- and SP+/+ mice. Integrated analysis revealed that the accelerated growth of ID8 tumors in SP-/- mice was associated with metabolic programming of cancer cells with up-regulation of the genes involved in glycolysis, fatty acid oxidation, and oxidative phosphorylation. Tumors also exhibited perturbed redox homeostasis, as well as mitochondrial and ribosomal biogenesis. Comparative analysis of the syngeneic tumors with human high grade serous ovarian cancer (HGSC) revealed upregulation of the same genes involved in metabolic programming with inverse correlation with SPARC transcript expression, implying a role of stromal-SPARC in the metabolic programming of HGSC. Bio-energetic studies revealed that SPARC inhibits basal glycolysis, glycolytic reserve, oxygen consumption rate and mitochondrial ATP synthesis. To the best of our knowledge this is the first study that characterizes the metabolic programming of the ovarian cancer ecosystem by host-SPARC.
Citation Format: Christine Naczki, Bincy John, Chirayu Patel, Ashlyn Lafferty, Alia Ghoneum, Hesham Afify, Amanda Davis, Guangxu Jin, Neveen Said. Integrated molecular analysis reveals novel SPARC-regulated metabolic programming in ovarian cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1448.
Abstract
Fractionated partial or whole brain irradiation (fWBI) is often required to treat metastatic brain cancer. Unfortunately, fWBI can lead to the development of a progressive, irreversible ...cognitive impairment in patients who survive longer than six months following treatment. Prior studies suggest that neuroinflammation and alterations in neuronal plasticity may play a role in this treatment-related side effect. In the current study we determined that radiation-induced inflammatory activation of primary rat cortical astrocytes results in downregulation of mRNA and protein for the GLT-1 glutamate transporter, which functions to regulate glutamate signaling at excitatory synapses. Furthermore, we show that delivery of clinically-relevant fWBI (40Gy, 5Gy/fraction, 2x/week) to young adult male rats results in 51% decrease in Glt-1 mRNA in the cortex at 48 hours. Two months after completion of fWBI, cortical GLT-1 mRNA is further decreased to 25% the levels observed in sham controls, while at six months following fWBI GLT-1 levels remain 43% decreased compared to controls. We have previously identified two classes of compounds, the renin-angiotensin system inhibitors and PPAR agonists, which ameliorate the fWBI-induced cognitive impairment in this rodent model, although the full mechanisms by which these compounds protect cognition remain unknown. We found that dietary administration of the angiotensin receptor blocker L-158-809, but not the ACE inhibitor Ramipril, prevents fWBI-induced loss of cortical GLT-1 mRNA at 48 hours, while both cognition-sparing compounds restored GLT-1 expression at 2 months post-fWBI. Similarly, administration of the PPARδ agonist GW0742 prevented the fWBI-induced decrease of GLT-1 mRNA observed 6 months post-fWBI. Together these data suggest that radiation-induced downregulation of GLT-1 is a potential mechanism contributing to the development of radiation-induced brain injury, including cognitive impairment, and that GLT-1 may represent a druggable target to prevent this treatment-related cognitive decline. (Supported by NIH CA112593)
Citation Format: Mitra Kooshki, Christine Naczki, Michael E. Robbins, Linda J. Metheny-Barlow. Radiation-induced downregulation of GLT-1 glutamate transporter mRNA expression is reversed by renin-angiotensin system inhibitors. 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 1792. doi:10.1158/1538-7445.AM2015-1792
Proper functional association between mural cells and endothelial cells (EC) causes EC of blood vessels to become quiescent. Mural cells on tumor vessels exhibit decreased attachment to EC, which ...allows vessels to be unstable and proliferative. The mechanisms by which tumors prevent proper association between mural cells and EC are not well understood. Since gap junctions (GJ) play an important role in cell-cell contact and communication, we investigated whether loss of GJ plays a role in tumor-induced mural cell dissociation.
Mural cell regulation of endothelial proliferation was assessed by direct co-culture assays of fluorescently labeled cells quantified by flow cytometry or plate reader. Gap junction function was assessed by parachute assay. Connexin 43 (Cx43) protein in mural cells exposed to conditioned media from cancer cells was assessed by Western and confocal microscopy; mRNA levels were assessed by quantitative real-time PCR. Expression vectors or siRNA were utilized to overexpress or knock down Cx43. Tumor growth and angiogenesis was assessed in mouse hosts deficient for Cx43.
Using parachute dye transfer assay, we demonstrate that media conditioned by MDA-MB-231 breast cancer cells diminishes GJ communication between mural cells (vascular smooth muscle cells, vSMC) and EC. Both protein and mRNA of the GJ component Connexin 43 (Cx43) are downregulated in mural cells by tumor-conditioned media; media from non-tumorigenic MCF10A cells had no effect. Loss of GJ communication by Cx43 siRNA knockdown, treatment with blocking peptide, or exposure to tumor-conditioned media diminishes the ability of mural cells to inhibit EC proliferation in co-culture assays, while overexpression of Cx43 in vSMC restores GJ and endothelial inhibition. Breast tumor cells implanted into mice heterozygous for Cx43 show no changes in tumor growth, but exhibit significantly increased tumor vascularization determined by CD31 staining, along with decreased mural cell support detected by NG2 staining.
Our data indicate that i) functional Cx43 is required for mural cell-induced endothelial quiescence, and ii) downregulation of Cx43 GJ by tumors frees endothelium to respond to angiogenic cues. These data define a novel and important role for maintained Cx43 function in regulation of vessel quiescence, and suggest its loss may contribute to pathological tumor angiogenesis.
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