bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A60: DOK1 gene may act as a tumor suppressor in breast cancer
Authors: Buyru Nur , Tuna Esin , Ozdemir Filiz , Bulut Pelin , Dalay Nejat
Published: May 2016

The DOK1 (Downstream of Tyrosine kinase 1) gene is a putative tumor suppressor gene located on human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. The DOK1 protein is a member of the Dok protein family comprising seven members. It inhibits MAP kinase activity and displays anti-proliferative activities. The DOK1 gene may act as a tumor suppressor gene in ovarian cancer, Burkitt lymphoma, head and neck cancer (HNC), chronic lymphocytic leukaemia (CLL) and lung cancer by inhibiting cell proliferation. So far there is no study in the literature analyzing the relationship between the DOK1 gene and breast cancer. The aim of this study was to investigate the role of the DOK1 gene in breast cancer.

The tumors and adjacent normal tissues were collected from 50 patients who underwent surgery for breast cancer at the Bezmialem Vakif University Medical Faculty. For expression analysis, after isolation of RNA cDNA was been obtained. Gene expression levels in the tumors and adjacent normal tissues were determined by quantitative RT-PCR using the 2-ΔΔCT method. The results were normalized using the G6PD gene as reference. For mutation analysis, DNA was isolated and analyzed by cycle sequencing to detect mutations. The SPSS 21.0 software package was used for statistical analysis. P<0.05 was considered significant.

When the DOK1 expression levels in the tumors and adjacent normal tissues were compared we observed that DOK1 expression was decreased in 31 (62%) patients and increased in 16 (32%) patients. The decrease of DOK1 expression was associated with age (p=0.031), menopausal status (p=0.017), histologic type, (p=0.015) and stage (p=0.017).

We identified 11 different alterations in the non-coding regions of the gene. One of these alterations was in the 5'-UTR and the remaining 10 were located in the introns 1 and 4. We did not observe any significant association between the mutations and expression level of the DOK1 gene. Our results, suggest that the DOK1 gene might act as a tumor suppressor in breast cancer.

Citation Format: Nur Buyru, Esin Tuna, Filiz Ozdemir, Pelin Bulut, Nejat Dalay. DOK1 gene may act as a tumor suppressor in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A60.
Abstract B22: PSMD2 is a molecular marker for a poor prognosis and determines cancer stem cells traits in breast cancer
Authors: Jung So-Youn , Jo Seolwha , Kang Eunji , Noh Dong-Young , Han Wonshik
Published: May 2016

The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSCs that is also capable of differentiating into non-self -renewing cell populations that constitute the bulk tumor mass. These cells have stem cell characteristics and may play a role in tumorigenic growth, metastasis, and therapeutic resistance. Here we interrogated the oncogenic roles of PSMD2, a subunit of the 19S regulatory complex of proteasomes, as a constituent of a signature is a candidate oncogene in breast cancer. PSMD2 is required for breast CSCs proliferation both in vitro and in vivo, as shRNA-mediated PSMD2 silencing attenuated tumor growth. Further, PMSD2 is sufficient to induce stem cell characteristics, as forced PSMD2 expression facilitated mammosphere formation and increased the CD44+ breast CSC-like population. Mechanistically, the action of PSMD2 for induction of breast stemness is mediated by transcriptional regulation of the Notch signaling pathway and PSMD2 inhibition were associated with changes in epithelial-mesenchymal transition (EMT) phenotype. Knockdown of PSMD2 induced dramatic morphological changes, the typical spindle-like appearance cells was replaced with the round shape, like epithelial morphology. Moreover, treatment of TGF-β, the main inducer of EMT, lead to increased expression of PSMD2. PSMD2 silencing up-regulated E-cadherin and down-regulated expression of vimentin, N-cadherin and snail, mesenchymal markers. Clinically, PSMD2 expression was elevated in basal-like TNBC (triple negative breast cancer), correlated with poor prognosis, insilico analysis on a dataset comprising over 2413 microarrays of the web application bc-GenExMiner. Altogether, our findings suggest that PSMD2 may be a good molecular target candidate and PSMD2 inhibitor may kills breast CSCs and ameliorates poor prognosis of breast cancer.

Note: This abstract was not presented at the conference.

Citation Format: Kyung-Min Lee, So-Youn Jung, Seolwha Jo, Eunji Kang, Dong-Young Noh, Wonshik Han. PSMD2 is a molecular marker for a poor prognosis and determines cancer stem cells traits in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B22.
Abstract B28: Activating mutations in PIK3CB confer resistance to PI3K inhibition in PTEN-deficient breast cancer and define a novel oncogenic role for p110β
Authors: Nakanishi Yoshito , Walter Kimberly M. , Spoerke Jill M. , O'Brien Carol , Huw Ling Y. , Hampton Garret M. , Lackner Mark R.
Published: May 2016

Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs commonly in breast cancers via mechanisms that include loss of function of the tumor suppressor phosphatase and tensin homolog (PTEN). Experience with other successful targeted agents suggests that clinical resistance to PI3K inhibitors is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN deficient breast cancers. PTEN null breast cancer cell lines were selected for resistance to a pan-PI3K inhibitor, GDC-0941. Comprehensive molecular and cellular profiling was conducted to identify the mechanism of resistance. We generated GDC-0941 resistant derivatives of PTEN deficient EVSA-T and ZR-75-1 cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines. We found that the PIK3CB mutation at D1067 position was recurrent in cancer patients. Stable expression of mutant PIK3CB variants in PTEN null breast cancer cells conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. We showed further that the p110β D1067Y mutant is highly activated and elevates PIP3 level at the cell membrane, promoting localization and activation of AKT and PDK1 at the cell membrane and driving PI3K signaling to a level that can overcome treatment with proximal inhibitors. Finally, we show that the PIK3CB D1067Y mutant can behave as an oncogene and transform normal cells, and that PTEN knock down enhances this activity. These novel preclinical and clinical findings implicate PIK3CB D1067 alterations as a novel oncogene that may cause resistance to selective PI3K inhibitor treatment. Taken together with previous findings that PTEN deficient cancers tend to signal through p110β rather than p110α, this work also suggests PTEN deficient breast cancers may depend on p110β and are thereby susceptible to PIK3CB mutation as an escape mechanism from PI3K inhibition.

Citation Format: Yoshito Nakanishi, Kimberly M. Walter, Jill M. Spoerke, Carol O'Brien, Ling Y. Huw, Garret M. Hampton, Mark R. Lackner. Activating mutations in PIK3CB confer resistance to PI3K inhibition in PTEN-deficient breast cancer and define a novel oncogenic role for p110β. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B28.
Abstract PR02: The spliceosome is a therapeutic vulnerability in MYC-driven breast cancer
Authors: Hsu Tiffany , Simon Lukas , Neill Nicholas , marcotte richard , Sayad Azin , Karlin Kristen , Lagisetti Chandraiah , Cooper Thomas , Webb Thomas , Neel Benjamin , Shaw Chad , Westbrook Thomas (“Trey”)
Published: May 2016

c-MYC (MYC) hyperactivation is one of the most common drivers of human breast cancer and correlates with poor prognosis. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. Like other classic oncogenes, hyperactivation of MYC leads to collateral stresses onto breast cancer cells, suggesting that tumors harbor unique vulnerabilities arising from oncogenic activation of MYC. Herein, we discover the spliceosome as a new target of oncogenic stress in MYC-driven cancers. We demonstrate that core components of the spliceosome and its catalytic activity are required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces global changes in mRNA metabolism and increases the burden on the core spliceosome to process pre-mRNA. In primary human breast cancers, MYC hyperactivation is associated with altered splicing efficiency. In contrast to normal mammary epithelium, partial inhibition of the spliceosome in MYC-hyperactivated breast cancers leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of essential cell processes. Importantly, genetic or pharmacologic inhibition of the spliceosome in vivo impairs survival, tumorigenicity, and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing and that components of the spliceosome are therapeutic entry points for aggressive MYC-driven breast cancers.

Citation Format: Tiffany Hsu, Lukas Simon, Nicholas Neill, richard marcotte, Azin Sayad, Kristen Karlin, Chandraiah Lagisetti, Thomas Cooper, Thomas Webb, Benjamin Neel, Chad Shaw, Thomas (“Trey”) Westbrook. The spliceosome is a therapeutic vulnerability in MYC-driven breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR02.
Abstract A02: A kinome-wide RNAi screen in Drosophila glia and human GBM models reveals Stk17A drives neoplastic glial proliferation and migration
Authors: Wardwell-Ozgo Joanna , Mosley Colleen , Kornblum Harley , Read Renee
Published: May 2016

Glioblastoma multiforme (GBM), the most common primary malignant brain tumor, are highly proliferative, diffusely invasive, and incurable by current therapies. Genetic and molecular analyses reveal that GBMs frequently harbor activating mutations in the EGFR receptor tyrosine kinase (EGFR) and Pi-3 kinase (PI3K) signaling pathways. While the ability of these mutations to drive gliomagenesis has been verified in mouse models, current data also indicate that EGFR and PI3K signaling cooperates with as yet unknown factors to drive its invasive pathology and therapeutic resistance in GBM. To identify such factors, we performed a cross-species, multidisciplinary genetic screen in both Drosophila melanogaster and mammalian GBM model systems designed to identify novel genes that augment EGFR- and PI3K- dependent neoplasia.

Our results in both Drosophila and GBM cell culture and animal model systems indicate that Stk17A, a cytoplasmic serine-threonine kinase subject to copy-gain and overexpression in GBM, is necessary for GBM cell proliferation and invasion. Our data also illustrate that overexpression of Stk17A in combination with oncogenic EGFR mutations is sufficient to promote increased glial cell proliferation and invasion. Furthermore, in our GBM model systems, we find that Stk17A overexpression stimulates increased phosphorylation and activity of non-muscle myosin II regulatory light chain (MRLC), a known Stk17A substrate and key regulator of the cell cycle and cellular motility, which itself is required for neoplastic glial transformation. Finally, oncogenomic analysis of GBMs and other high-grade gliomas reveal that Stk17A becomes overexpressed in association with EGFR-PI3K mutations, and Stk17A copy-gain and overexpression are significantly associated with poor prognosis in patients.

Together, these mechanistic insights indicate that Stk17A potentiates the oncogenenic effects of EGFR- and PI3K-driven tumors through the upregulation of MLRC activity, and suggest that Stk17A may serve as a novel therapeutic target for EGFR-PI3K dependent GBMs and high-grade gliomas. Current work is continuing to examine the molecular function of Stk17A in the context of GBM, normal glia, and neural stem cells in order to further our understanding of how Stk17A contributes to tumor pathology.

Citation Format: Joanna Wardwell-Ozgo, Colleen Mosley, Harley Kornblum, Renee Read. A kinome-wide RNAi screen in Drosophila glia and human GBM models reveals Stk17A drives neoplastic glial proliferation and migration. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A02.
Abstract B22: Analysis of Lgr5-positive cells in mouse mammary gland
Authors: Zhang Lixing , Howe Louise , Kolesnick Richard , Brown Anthony
Published: May 2016

Lgr5 has been identified as a stem cell marker in multiple adult tissues, and individual Lgr5+ mammary epithelial cells are capable of regenerating mouse mammary glands in transplantation assays. Here we investigated the proliferation and differentiation capabilities of murine mammary Lgr5+ cells in vitro, and their distribution in mouse mammary glands in vivo. Equal numbers of sorted Lgr5+ and Lgr5- epithelial cells were cultured in Matrigel to evaluate their stem-like properties in vitro. Compared with Lgr5- cells, Lgr5+ cells were more efficient at generating organoids in 3D culture, and the Lgr5+ cell-derived organoids grew to a larger size. Moreover, Lgr5+ cell-derived organoids survived repeated passaging and contained cells of both luminal and basal lineages, as indicated by keratin marker expression. Incidence of Lgr5+ cells in organoids was confirmed by in situ hybridization. These studies indicate that Lgr5+ cells retain the self-renewal and differentiation capabilities of stem cells ex vivo. The in vivo distribution of Lgr5+ cells was investigated using X-Gal-stained mammary glands from Lgr5-lacZ reporter mice ranging from 5 to 32 weeks old. In both adolescent and adult mice, we observed a graded distribution of Lgr5+ cells with greater abundance in the nipple-proximal (ventral) ducts than in the lymph-node proximal central region, and fewest Lgr5+ cells in the distal (dorsal) region of the glands. Lgr5+ cells from all mammary gland regions appeared capable of yielding organoids in culture. These results corroborate in organoid culture the known stem-like properties of Lgr5+ cells defined in intact mouse mammary gland.

[Supported by NIH grant CA186981 and the China Scholarship Council]

Citation Format: Lixing Zhang, Louise Howe, Richard Kolesnick, Anthony Brown. Analysis of Lgr5-positive cells in mouse mammary gland. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B22.
Abstract B30: Regulation of proliferation and differentiation by atonal homolog 1 phosphorylation
Authors: Tomic Goran , Winton Douglas J. , Philpott Anna
Published: May 2016

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix (bHLH) transcription factor that acts as a tumor-suppressor as well as drives the differentiation of intestinal progenitor cells. Phosphorylation of Atoh1 is an important regulatory mechanism of its activity; proteins of the bHLH family can be phosphorylated by cyclin-dependent kinases during the cell cycle, which affects their differentiation-driving potential.

We generated a colon cancer cell line model to investigate site-specific phosphoregulation of Atoh1. A mutant Atoh1, which has multiple serine/threonine residues changed to alanine at serine-proline (SP) and threonine-proline (TP) sites (9S/T-A Atoh1), was generated and expressed in a doxycycline-inducible model at a single copy level. The loss of phosphorylation induced hyper-activation of Atoh1 with respect to differentiation and proliferation arrest; 9S/T-A Atoh1 inhibited proliferation more than the wild-type protein. Overexpression of Atoh1 caused G1 cell-cycle arrest. Atoh1 induced expression of the secretory lineage markers, while absorptive lineage markers were down regulated. Phospho-mutant protein was able to not only inhibit proliferation but also drive the differentiation of previously undifferentiated colon cancer cell line more strongly than the wild type protein.

Investigating the significance of phosphorylation of Atoh1 in the context of its tumor-suppressive role is of great importance in colon cancer. A better insight into the differentiation processes and their deregulation in cancer has the potential to open further avenues and introduce promising strategies in differentiation-targeted cancer therapies.

Citation Format: Goran Tomic, Douglas J. Winton, Anna Philpott. Regulation of proliferation and differentiation by atonal homolog 1 phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B30.
Abstract IA17: Dynamic changes in the epigenetic landscape during retinal development, cellular reprogramming, and tumorigenesis
Authors: Dyer Michael
Published: May 2016

The stepwise progression from proliferating multipotent progenitor cells to terminally differentiated neurons in the developing central nervous system (CNS) is marked by dramatic changes in gene expression programs. Those changes in gene expression are accompanied by changes in histone modifications at individual promoters, gene bodies and enhancers as well as changes in the higher order structure of the chromatin. Once established during development, the cell-type specific epigenome of differentiated cells is thought to be relatively stable. Indeed, one of the major barriers to reprogramming differentiated cells such as neurons into induced pluripotent stem cells (iPSCs) is the reversal of the epigenetic landscape that was established during differentiation. For some cell types, the cell-type specific epigenetic programs are not completely erased during reprogramming and the resulting induced pluripotent stem cells (iPSCs) retain an epigenetic memory of their cellular origins. The dynamic changes in the epigenome that occur during development are also important for human disease. Childhood cancers are developmental tumors that arise during critical periods of development across diverse tissues. In a childhood cancer of the developing retina called retinoblastoma, it was shown the epigenetic reprogramming is required for tumorigenesis and this led to the identification of a novel druggable target for this ocular tumor. However, it is not known if the epigenetic changes that accompany tumorigenesis in childhood cancer are similar to those that occur during cellular reprogramming or if it is a completely distinct process that is required for oncogenic transformation. In this presentation, I will compare and contrast the changes in the epigenome that accompany changes in gene expression during retinal development, retinal neuron reprogramming and in retinoblastoma to gain a deeper understanding of the epigenome in development and cancer.

Citation Format: Michael Dyer. Dynamic changes in the epigenetic landscape during retinal development, cellular reprogramming, and tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA17.
Abstract IA31: Control of TGF-β responsiveness and epithelial plasticity
Authors: Derynck Rik , Budi Erine , Muthusamy Baby-Periyanayaki , Katsuno Yoko , Du Dan
Published: May 2016

TGF-β signaling inhibits cell growth in epithelial cells and thus acts as tumor suppressor, yet increased TGF-β signaling often promotes cancer progression through effects on the tumor micro-environment and effects on the carcinoma cells that can lead to epithelial plasticity responses and increased invasion. The responsive epithelial or carcinoma cells can regulate their cell surface levels of TGF-β receptors, and thus enhance their responses to TGF-β. In addition to transcription control of TGF-β receptor expression, the cells can rapidly mobilize TβRII and TβRI from a large intracellular receptor pool to the cell surface, and thus enhance their TGF-β responsiveness. For example, high glucose, as seen in hyperglycemia, rapidly induces high levels of cell surface receptors that increase the cell’s TGF-β responsiveness, resulting in extracellular matrix production and increased cell size. Similarly, increased cell surface TGF-β receptor levels and TGF-β responsiveness also occur in response to insulin, which is therapeutically used against diabetes-associated hyperglycemia. The insulin-induced increase in cell surface levels requires activation of Akt, which then phosphorylates the membrane-associated RabGAP AS160, thus alleviating AS160-mediated retention of vesicles containing TβRII and TβRI, and allowing them to reach the cell surface. Additionally, the levels of functional TGF-β receptor complexes at the cell surface is controlled by TACE-mediated ectodomain shedding, which is activated in response to Erk and p38 MAPK activation. We will discuss the physiological implications of these multiple levels of control of TGF-β receptor cell surface presentation and TGF-β responsiveness in cancer cell behavior and cancer progression. Finally, the epithelial plasticity response to TGF-β is also controlled by the direct interaction of activated Smad3 with a methyltransferase, which consequently controls the histone modifications of the gene encoding the transcription factor Snail, a master regulator that drives epithelial-mesenchymal transition. Thus, Smad-mediated changes of histone methylation complement direct Smad-mediated transcription effects in the control of epithelial-mesenchymal transition.

Citation Format: Rik Derynck, Erine Budi, Baby-Periyanayaki Muthusamy, Yoko Katsuno, Dan Du. Control of TGF-β responsiveness and epithelial plasticity. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA31.
Abstract IA33: Regulation of TGF-β receptor signaling in cancer
Authors: Dijke Peter ten
Published: May 2016

TGF-β plays a crucial role during development and in maintaining tissue homeostasis. Perturbation in signaling via TGF-β type I and type II serine/threonine kinase receptors and intracellular SMAD transcriptional effectors, can lead to a large variety of diseases, including cancer. TGF-β has a dual role in cancer progression. During the early phase, TGF-β acts as a tumor suppressor, exemplified by deletions or mutations in the core components of the TGF-β signaling pathway. In late phase, TGF-β promotes processes that support tumor progression such as tumor cell invasion, dissemination, and immune evasion. Clinical trials for the treatment of cancer patients using systemic TGF-β signaling inhibitors have been initiated. These agents target TGF-β ligand or TGF-β receptor and inhibit all responses, and may have unwanted on-target effects. I will present our efforts to identify new druggable regulators of TGF-β signaling, which may allow for a more selective antagonism of the tumor promoting pathways by TGF-β. For example, E3 ubiquitin ligase TRAF4 and deubiquitinating enzyme USP4 - that are highly expressed in breast cancer - regulate the stability of specific TGF-β signaling components and thereby promote TGF-β-induced breast cancer cell invasion and metastasis.

Citation Format: Peter ten Dijke. Regulation of TGF-β receptor signaling in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA33.
Abstract PR01: 3D enhancer clusters in the response to Wnt-driven colorectal cancer
Authors: Schuijers Jurian , Day Daniel S. , Saint-Andre Violaine , Young Richard A.
Published: May 2016

Super-enhancers are clusters of enhancers that drive expression of genes key to development and cancer. We recently showed that super-enhancers and their target genes occur within DNA loops bounded by CTCF proteins. These chromosome structures constrain the activities of enhancers to genes that occur within the same loop, and were thus called insulated neighborhoods. Recent studies have shown that signaling pathways converge on super-enhancers, but the effect of signaling-mediated gene activation has yet to be investigated in the context of insulated neighborhood structure and function. We used a model of Wnt-pathway activation in colon cancer to investigate Wnt-mediated activation of target genes within insulated neighborhoods. When the Wnt-pathway was activated through genetic deletion of the tumor suppressor Apc in intestinal organoids, all enhancers within insulated neighborhoods that contained Tcf4 binding sites were activated, including enhancers that were not considered to contribute to super-enhancers. Thus, Wnt-stimulation co-activated multiple enhancers that are not in linear proximity, yet cluster in 3D space within insulated neighborhoods. We conclude that insulated neighborhoods allow the formation of 3D enhancer clusters whose constituents can operate concertedly, and thus expand the definition of super-enhancers to incorporate 3D chromatin organization.

This abstract is also presented as Poster A15.

Citation Format: Jurian Schuijers, Daniel S. Day, Violaine Saint-Andre, Richard A. Young. 3D enhancer clusters in the response to Wnt-driven colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR01.
Abstract A10: SLC7A11 contributes to the pathogenesis of lung cancer
Authors: Ji Xiangming Ji , Qian Jun , Rahman Jamshedur , Harris Brad , Hoeksema Megan , Chen Heidi , Eisenberg Rosana , Young Jamey
Published: May 2016

Introduction: Many tumors increase uptake and dependence on nutrients such as glucose, cysteine or glutamine. These basic observations on cancer cell metabolism have opened multiple new diagnostic and therapeutic avenues in cancer. Here, we investigated the function of SLC7A11 (xCT), a cystine/glutamate anti-porter, in the pathogenesis of lung cancer. Recently, we found xCT to be strongly overexpressed at the cell surface of lung cancers and began to investigate the mechanisms by which xCT may contribute to lung cancer progression.

Methods: The expression of xCT and its related genes in 411 patients with non small cell lung cancer (NSCLC) were evaluated in publically available lung cancer gene expression datasets. The association between xCT expression and overall survival, stage of diseases were tested in tissue microarray datasets. Gain of function studies for xCT were undertaken in the normal airway epithelium cell lines (BEAS2B and 16HBE) to investigate the role of xCT in regulating cell metabolism, growth and survival in normal and malignant lung epithelial cells.

Results: Our results shows that xCT proein is overexpressed in 38% of NSCLC patients. We also found that expression of xCT mRNA is strongly associated with disease stage (p<0.01), smoking history (p =0.0202) and overrall survival (p<0.01). Overexpression of xCT in normal airway epithelium cells (16HBE and BEAS2B) induces the colony formaion in soft agar and tumor formation in nude mice. Targeting these cells with xCT overexpression by sulfasalazine significantly reduced tumor burden in mice by 50%. Furthermore, the overexpresion of xCT promotes increased glucose consumption, glutamine consumption and lactate production.

Conclusion: Our results demonstrate that xCT is highly expressed in a subset of NSCLCs. The induction of xCT in transformed aiway epithelial cells causes tumor formation in nude mice. The mechanism of malignant transformation involves the Warburg effect. Further investigation into the mechanisms of transformation may help in answering fundamental questions pertaining to how glutamate/cystine exchange contributes to lung cancer development and progression and also in establishing xCT as a potential novel therapeutic target in lung cancer.

Citation Format: Xiangming Ji Ji, Jun Qian, Jamshedur Rahman, Brad Harris, Megan Hoeksema, Heidi Chen, Rosana Eisenberg, Jamey Young. SLC7A11 contributes to the pathogenesis of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A10.
Abstract A22: An LKB1-CRTC1 circuit regulates glycosylated COX-2 and predicts drug response in lung cancer
Authors: Cao Chunxia , Gao Ruli , Zhang Min , Amelio Antonio L. , Fallahi Mohammad , Chen Zirong , Gu Yumei , Hu Chengbin , Welsh Eric A. , Engel Brienne E. , Haura Eric , Cress W. Douglas , Wu Lizi , Zajac-Kaye Maria , Kaye Frederic J.
Published: May 2016

Cyclooxygenase-2 (COX-2) directs the synthesis of prostaglandins important for mitogenic signaling. Here we report that COX-2 is a transcriptional target of the CREB co-activator CRTC1. In addition, we detected a correlation between the LKB1-null status and presence of 72/74 kDa glycosylated COX-2, but not inactive hypoglycosylated COX-2 in fresh lung adenocarcinoma samples. Since CRTC1 is suppressed by cytoplasmic shuttling following LKB1/AMPK/SIK phosphorylation, we developed an LKB1 signature in lung cancer to search the Connectivity-MAP drug response database. Remarkably, all high-ranking drugs positively associated with the LKB1-null signature were known CRTC1 activators. Somatic LKB1 mutations are present in 20% of lung adenocarcinomas and we observed growth and cell motility inhibition with COX-2 inhibitors in LKB1-null lung cancer cells with activated CRTC1, but negligible inhibition in LKB1-wildtype cells. In summary, the CREB co-activator CRTC family directly links LKB1 with COX-2 activation and provides a new framework for selecting patients for COX-2 inhibition.

Citation Format: Chunxia Cao, Ruli Gao, Min Zhang, Antonio L. Amelio, Mohammad Fallahi, Zirong Chen, Yumei Gu, Chengbin Hu, Eric A. Welsh, Brienne E. Engel, Eric Haura, W. Douglas Cress, Lizi Wu, Maria Zajac-Kaye, Frederic J. Kaye. An LKB1-CRTC1 circuit regulates glycosylated COX-2 and predicts drug response in lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A22.
Abstract A36: Amino acids rather than glucose accounts for the majority of cell mass in rapidly proliferating mammalian cells
Authors: Hosios Aaron M. , Hecht Vivian C. , Johnson Marc , Rathmell Jeffrey C. , Manalis Scott R. , Heiden Matthew G. Vander
Published: May 2016

To facilitate growth and division, proliferating cells must duplicate their mass over the cell cycle. Glucose and glutamine are the most consumed nutrients by proliferating mammalian cells, but the extent to which these and other nutrients contribute directly to cell mass has not been investigated. Using isotope labeled nutrients, we quantified the fraction of cell mass derived from different fuels and find that surprisingly neither glucose nor glutamine provide the majority of carbon present in cells. Instead, amino acids, which are consumed at substantially lower rates, together account for the majority of cell mass. While glucose carbon has diverse fates, glutamine is overwhelmingly diverted into protein and its incorporation into cell mass greatly exceeds that of other amino acids, suggesting that glutamine-driven anapleurosis is primarily used for amino acid biosynthesis. Our findings demonstrate that the most heavily consumed nutrients do not make a proportionately large contribution of carbon to macromolecule biosynthesis.

Citation Format: Aaron M. Hosios, Vivian C. Hecht, Marc Johnson, Jeffrey C. Rathmell, Scott R. Manalis, Matthew G. Vander Heiden. Amino acids rather than glucose accounts for the majority of cell mass in rapidly proliferating mammalian cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A36.
Abstract A43: BRAF-mutated melanomas exhibit distinct metabolic programs that may determine therapeutic response
Authors: Hardeman Keisha N. , Peng Chengwei , Paudel Bishal , Tyson Darren , Young Jamey D. , Quaranta Vito , Fessel Joshua P.
Published: May 2016

Nearly 60% of melanomas have an activating mutation in the BRAF kinase. Targeted inhibition of mutant BRAF has prolonged overall survival, but responses are highly variable. To investigate the response variability, we utilized a panel of eight BRAF V600E or D mutated melanoma cell lines and treated them with the targeted mutant BRAF inhibitor, PLX4720. Under PLX4720 treatment, the cell lines exhibited a spectrum of concentration-dependent sensitivities based on measured proliferative rates over 96 hours of treatment. Since dysregulated metabolism has been shown to influence therapy resistance, we hypothesized that this spectrum of sensitivity would vary concordantly along a single metabolic phenotype. To test this hypothesis we measured glucose and amino acid uptake, lactate production, extracellular acidification upon mitochondrial inhibition, and proliferative rates under lactogenic glycolysis (oxamic acid) and ATP-synthase (oligomycin) inhibition in the cell lines. Interestingly, we did not find a single metabolic spectrum but rather unique clusters of metabolic phenotypes ranging from glycolysis-, amino-acid-, and oxidative phosphorylation-dependent to high metabolically flexible cell lines. Particularly, some cell lines were extremely sensitive to even low concentrations of oligomycin, whereas the majority of cell lines exhibited concentration-independent decreases of approximately ½ their proliferative rate under vehicle control. Metabolism and cell growth are intimately linked to cell cycle status, so we asked whether sensitivity to PLX4720 is abrogated when cells are selected under PLX4720 treatment. Using a fluorescence ubiquitination cell cycle indicator (FUCCI) to label dividing cells (G2/S/M phases), we pre-treated melanomas with PLX4720 or vehicle, flow-sorted FUCCI+ and FUCCI-cells, then re-treated with PLX4720. This revealed there was no positive selection for PLX4720-resistant subclones, as the PLX4720 treated FUCCI+/- cells pheno-copied the proliferative rates of the vehicle treated FUCCI+/- cells. Taken together, our results suggest that BRAF-mutated melanomas exist in a metabolically rich landscape that may be an important factor in response to targeted BRAF inhibition. The long-term goal is to translate these metabolic differences clinically, possibly by coupling metabolic and oncogene targeted therapies.

Citation Format: Keisha N. Hardeman, Chengwei Peng, Bishal Paudel, Darren Tyson, Jamey D. Young, Vito Quaranta, Joshua P. Fessel. BRAF-mutated melanomas exhibit distinct metabolic programs that may determine therapeutic response. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A43.
Abstract A47: The regulation of TIGAR
Authors: Lee Pearl , Cheung Eric C. , Vousden Karen H.
Published: May 2016

The p53 tumor suppressor protein inhibits the development of cancer by initiating various cellular responses including apoptosis, senescence and cell cycle arrest. In addition to this, recent studies have found that p53 is also able to influence cell metabolism. TP53-induced glycolysis and apoptosis regulator (TIGAR) was discovered as a p53 target functioning as a fructose-2,6-bisphosphatase. In this way, TIGAR can modulate glucose metabolism, resulting in enhanced NADPH production and antioxidant defence.

While discovered as a p53 target, basal expression of TIGAR is not affected by loss of p53 or its family member TAp73 in mice and increased expression of TIGAR in the small intestines is still observed following irradiation in these animals. Moreover, this induction is retained in mice deleted of both p53 and TAp73, demonstrating that TIGAR expression is not dependent on either of these transcription factors.

In mouse intestines, TIGAR is important in repair following tissue injury and mice that are TIGAR-deficient show elevated levels of oxidative stress following damage. As the Wnt/Myc signalling pathway is important in cellular proliferation and tissue regeneration, its potential role in regulating TIGAR was investigated. Deletion of APC in mouse intestines, leading to high Wnt pathway activation, results in an increased expression of TIGAR, suggesting that the Wnt/Myc signalling pathway may influence the regulation of TIGAR.

Together, the results suggest that TIGAR can be regulated through p53-dependent and independent mechanisms to contribute to the control of tissue regeneration and tumorigenesis.

Funded by Cancer Research UK, PL is a recipient of an MRC studentship.

Citation Format: Pearl Lee, Eric C. Cheung, Karen H. Vousden. The regulation of TIGAR. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A47.
Abstract A51: Targeting mTOR metabolism for glioblastoma cancer therapy
Authors: Tandle Anita , Kramp Tamalee , Tofilon Philip , Camphausen Kevin
Published: May 2016

Aerobic glycolysis (the Warburg effect), first recognized almost a century ago, by Otto Warburg is a core hallmark of cancer. The Warburg effect describes a switch in glucose metabolism from oxidative phosphorylation to glycolysis. Recently links have been established between the oncogenic pathways that drive tumorigenesis and the mechanistic basis of tumor cell metabolism. The kinase mTOR is a major driver of tumor metabolism and proliferation of cancer cells, acts downstream of numerous oncogenic pathways. Several drugs targeting the mTOR pathway are being developed, however the most common drug rapamycin does not inhibit mTOR complex-2. Therefore in the current study, we examined the potential benefit of MLN0128, a novel potent mTOR ATP competitive inhibitor, as a therapeutic strategy for glioblastoma (GBM), one of the deadliest brain tumors.

We studied the action of MLN0128 in human GBM tumor cells and patient derived stem cells. We show the inhibitory effect of MLN0128 in nanomolar concentrations on tumor proliferation in multiple GBM cell lines via the blockage of phospho-AKT, -ribosomal protein, -4EBP1 inhibiting both mTORC1 and mTORC2 signaling in GBM. Reduction of invasive potential and disruption of paxillin localization to focal adhesions was observed in vitro. MLN0128 treatment decreased glucose consumption and lactate production. It inhibited rate of glycolysis, glycolytic capacity and glycolytic reserve. It also inhibited ATP production, mitochondrial respiration and spare respiratory capacity. MLN0128 was superior in inhibiting metabolism compared to rapamycin. On clonogenic survival assay MLN0128 decreased GBM cell survival and increased radiosensitivity. In vivo treatment of mouse xenografts with MLN0128 downregulated mTOR targets and significantly inhibited tumor volume compared to drug and irradiation alone. Combination treatment of GBM cells with MLN0128 and other chemotherapeutic agents like carboplatin, irinotecan and TMZ showed synergistic effects when combination index was calculated in GBM tumor cells and stem cells. However, the therapeutic efficacy of TMZ combination was greater than MLN0128 combination with either carboplatin or irinotecan. Moreover, similar TMZ chemosensitivity was observed in MGMT non-methylated, TMZ in-sensitive GBM stem cells and MGMT methylated GBM cells. These findings support the rationale for clinical testing of MLN0128 in patients with GBM to provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors with standard care.

Citation Format: Anita Tandle, Tamalee Kramp, Philip Tofilon, Kevin Camphausen. Targeting mTOR metabolism for glioblastoma cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A51.
Abstract A60: Inhibition of growth of bladder cancer cells by compounds that affect glucose metabolism
Authors: Lea Michael A. , Altayyar Mansour , desBordes Charles
Published: May 2016

The tendency of cancer cells to have enhanced rates of glycolysis presents a target for combination therapy with agents that affect glucose metabolism. We have explored the action of three compounds on a panel of eight human bladder cancer cell lines showing a spectrum of growth rates. The compounds were an inhibitor of PFKFB3, namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), an inhibitor of pyruvate dehydrogenase kinase, dichloroacetate, and, paradoxically, phenformin. The biguanide phenformin increases glucose metabolism in some cancer cell lines but it can also be growth inhibitory. Effects on glucose metabolism and lactic acid production were monitored respectively by an enzyme-linked colorimetric assay for glucose in the medium and changes in the pH of the medium reflected by the absorbance of phenol red at 560 nm. Changes in these parameters were most marked for the rapidly growing T24 and UM-UC-3 bladder cancer cells and generally showed parallel changes with control and treated cells. We had previously established growth inhibitory effects of 10µM 3PO on the eight bladder cancer cell lines monitored by staining with sulforhodamine B. Inhibitory effects of 2.5 mM dichloracetate have been observed with additive inhibitory effects when cells were treated with combinations of 3PO and dichloracetate. Effects of phenformin as a single agent were studied at 10, 25 and 50µM. Concentration-related increases in medium acidification and glucose uptake were most consistently seen with T24 cells. The effects with 50µM phenformin could be blocked by coincubation with 2.5 mM dichloracetate. On the other hand, additive inhibitory effects on growth may be observed with combinations of phenformin and dichloroacetate. Sensitivity to the actions of phenformin could not be simply related to growth rate. The T24 and UM-UC-3 cells have similar growth rates and had similar inhibitory effects on growth when treated with phenformin. However, medium acidification and glucose uptake were less affected in the UM-UC-3 cells. Although there are conditions in which phenformin appears to increase the Warburg effect as it relates to glucose metabolism, there can be additive growth inhibitory effects when bladder cancer cells are treated with phenformin in combination with agents that inhibit glucose metabolism.

Citation Format: Michael A. Lea, Mansour Altayyar, Charles desBordes. Inhibition of growth of bladder cancer cells by compounds that affect glucose metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A60.
Abstract A85: Concurrent targeting of glutamine and asparagine metabolism produces synergistic inhibition of tumor cell proliferation
Authors: Schramm Cicely L. , Xie Zhi , Kindt Erick , Li Wenlin , Wang Fang , Fantin Valeria R.
Published: May 2016

One of the best characterized examples of metabolic changes occurring in a wide range of cancer cells is dependence on the non-essential amino acid glutamine. We identified requirements for several other non-essential amino acids, including asparagine, across a panel of glutamine-dependent and –independent breast and lung cancer cell lines. Glutamine and asparagine metabolism are tightly linked. Therefore, we hypothesized that concurrent targeting of the metabolism of these two non-essential amino acids could cooperatively interfere with tumor cell growth. Treatment with a glutaminase inhibitor, BPTES, and concurrent enzymatic depletion of asparagine produced a synergistic antiproliferative effect in many, but not all, cancer cell lines. Mechanistic data suggest that metabolic circuitry gets co-opted to maintain amino acid pools following combined targeting of glutamine and asparagine metabolism, resulting in mitochondrial dysfunction and redox imbalance. Following combination treatment, alterations in mitochondrial metabolism and cystine influx were observed, but overall amino acid levels remained surprisingly stable. These metabolic changes were reflected in cellular functional readouts as measured by Seahorse analysis and measurement of ROS levels. Importantly, they were also linked to the synergistic antiproliferative phenotype. Our results provide new mechanistic insight into the interplay between glutamine and asparagine metabolism in cancer cells and suggest that dual targeting of metabolically linked non-essential amino acids may be promising as a therapeutic strategy.

Citation Format: Cicely L. Schramm, Zhi Xie, Erick Kindt, Wenlin Li, Fang Wang, Valeria R. Fantin. Concurrent targeting of glutamine and asparagine metabolism produces synergistic inhibition of tumor cell proliferation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A85.