bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C52: Induction of homologous recombination by DNA nicks
Authors: Vriend Lianne E.M. , Krawczyk Przemek M. , Jasin Maria
Published: August 2014

Homologous recombination (HR) is a major mechanism of DNA repair, such that mutations in HR genes are associated with tumor susceptibility. We have used a recently adapted system for genome engineering to study HR mechanisms in mammalian cells. Clustered regularly-interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems that specifically cleave nucleic acids of invading pathogens. The effector phase of CRISPR system in Staphylococcus pyogenes consists of two main components, the endonuclease Cas9 and RNA strands that dictate the recognition sequence cleaved by Cas9. The activity of Cas9 resides in 2 nucleolytic domains, HNH-like and RuvC1-like, such that mutating the active site of either of these domains turns Cas9 into a DNA nicking enzyme.

Here we applied the commonly used DR-GFP HR reporter to investigate the repair of both DNA double-strand breaks (DSBs) and nicks induced by Cas9 in human cells. We show that a DSB, introduced by wild-type (wt) Cas9 and a guide RNA (gRNA) directed to the I-SceI site of the DR-GFP reporter, induces HR at levels similar to the I-SceI endonuclease, demonstrating the robustness of the system and confirming the potential of the CRISPR system as a genome engineering tool. Cas9 with mutations in either of the nucleolytic domains also stimulates HR, albeit at much lower levels, suggesting that a nick can also be repaired by HR. Stimulation of HR by DNA nicks is not reduced by PARP inhibition and has the same genetic requirements as DSB-induced HR. Given that SSBs are induced by endogenous DNA metabolic processes as well as by many anti-cancer treatments, our results may advance the understanding of mechanisms responsible for repair of these abundant DNA lesions.

Citation Format: Lianne E.M. Vriend, Przemek M. Krawczyk, Maria Jasin. Induction of homologous recombination by DNA nicks. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C52.
Abstract C55: Investigating mechanisms of gastrointestinal polyposis and osteoblastic tumor formation induced by mesenchymal Lkb1 deficiency
Authors: Laajanen Kaisa , Ollila Saara , Wong Iris PL , Vaahtomeri Kari , Leone Gustavo , Viollet Benoit , Tomi Makela P.
Published: August 2014

Lkb1 has emerged as an important tumor suppressor gene in both sporadic and hereditary cancer. Inactivating germline mutations in the LKB1 gene underlie familial Peutz-Jeghers Syndrome (PJS), a cancer-prone disorder characterized by predisposition to gastrointestinal polyposis. Targeted disruption of Lkb1 in mesenchymal smooth muscle cells was sufficient to recapitulate PJS-type polyposis in mice (Katajisto et al, 2008) and Lkb1 heterozygous mice develop osteoblastic tumors (Robinson et al, 2008). To investigate the mechanism(s) by which Lkb1 loss in mesenchymal tissues drives tumorigenesis, we deleted Lkb1 using two mesenchymal deletors (Fsp1-Cre and Twist2-Cre) expressed in mesenchymal progenitors. Heterozygous deletion of Lkb1 with either Fsp1-Cre (Lkb1flox/+;Fsp1-Cre) or Twist2-Cre (Lkb1flox/+;Twist2-Cre) results in both polyposis and osteoblastic tumor. Homozygous Lkb1 deletion using Fsp1-Cre (Lkb1flox/flox;Fsp1-Cre) leads to rapid polyposis and osteoblastic tumors with complete penetrance, indicating that complete loss of Lkb1 is advantageous for tumorigenesis. AMPK, a key substrate of Lkb1 connected to several other tumor suppressors, has been proposed to mediate Lkb1 tumor suppressive function. However, homozygous deletion of both AMPKα1 and AMPKα2 using Fsp1-Cre does not lead to tumorigenesis. These results implicate other Lkb1 substrate kinases in tumor suppression and suggest Lkb1 loss in a common progenitor underlies mesenchymal tumorigenesis.

Katajisto P, et al., (2008) LKB1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis. Nat Genet, 40(4): p. 455-9

Robinson J, et al., (2008) Osteogenic tumours in Lkb1-deficient mice. Exp Mol Pathol, 85(3): p. 223-6

Citation Format: Kaisa Laajanen, Saara Ollila, Iris PL Wong, Kari Vaahtomeri, Gustavo Leone, Benoit Viollet, Makela P. Tomi. Investigating mechanisms of gastrointestinal polyposis and osteoblastic tumor formation induced by mesenchymal Lkb1 deficiency. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C55.
Abstract C58: The transglutaminase 2 (TG2) as a new target of p53
Authors: Yeo Shi Yun
Published: August 2014

The transglutaminase type 2 (TG2) is a multifunctional protein that has been found previously to be upregulated in lung and breast cancer cells in response to DNA damage. Since the tumor suppressor p53 stabilizes and accumulates in the nucleus upon DNA damage, we investigated the relationship of TG2 and p53 in this study. We demonstrated that TG2 is a downstream target of p53. Knockdown of p53 down-regulated the expression of TG2 mRNA and protein. Similarly, over-expression of p53 upregulated the level of TG2 under normal cell culture condition and also with doxorubicin treatment. More importantly, we found that loss of TG2 enables the transformation of primary mammary epithelial cells in a sensitized colony forming assay. In this system, we used primary human mammary epithelial cells (HMECs) that over-express hTERT, SV40 Small T and RasV12; these cells depend only on the loss of p53 activity for full transformation. Interestingly, we observed a synergy in colony formation when both p21 and TG2 were knock-down, and this effect is even more pronounced when there is a deprivation of growth factors. We discovered that insulin and EGF can overcome the effects of p53 in colony formation and the combined loss of TG2 and p21 can replace the absence of insulin in the transformation of HMECs, similar to that of p53 knock-down. This suggests that loss of TG2 potentiates insulin signaling that could be important in cell transformation. Indeed, we showed that loss of TG2 increase the phosphorylation of 4EBP1 at Thr37/46. Our findings indicate that TG2 functions like a tumor suppressor and is an important target of p53. The loss of TG2 results in cell transformation by mimicking insulin signaling.

Citation Format: Shi Yun Yeo. The transglutaminase 2 (TG2) as a new target of p53. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C58.
Abstract C59: Estradiol and mTORC2 orchestrate to enhance prostaglandin biosynthesis andtumorigenesis in tuberous sclerosis complex
Authors: Li Chenggang , Lee Po-Shun , Gu Xiaoxiao , Sun Yang , Zhang Erik , Csibi Alfredo , Li Jing , Priolo Carmen , Guo Yanan , Wu Chin-Lee , Blenis John , Xu Kai-Feng , Henske Elizabeth Petri , Levy Bruce , Kwiatkowski David , Yu Jane J.
Published: August 2014

Lymphangioleiomyomatosis (LAM) is a female predominant progressive and neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically due to TSC2 mutations resulting in mTORC1 activation in proliferative smooth muscle-like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was increased at baseline in TSC2-deficient cells, and was not affected by rapamycin treatment, but decreased by Torin 1 treatment and Rictor knockdown, associated with reduction of levels phospho-Akt (S473). Prostaglandin production was also increased in TSC2-deficient cells. In preclinical models, Celecoxib or aspirin treatment suppressed tumor progression. LAM patients had significantly higher

serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR-hyperactivation.

Citation Format: Chenggang Li, Po-Shun Lee, Xiaoxiao Gu, Yang Sun, Erik Zhang, Alfredo Csibi, Jing Li, Carmen Priolo, Yanan Guo, Chin-Lee Wu, John Blenis, Kai-Feng Xu, Elizabeth Petri Henske, Bruce Levy, David Kwiatkowski, Jane J. Yu. Estradiol and mTORC2 orchestrate to enhance prostaglandin biosynthesis andtumorigenesis in tuberous sclerosis complex. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C59.
Abstract C66: MUC1-C oncoprotein induces aldehyde dehydrogenase expression and activity in breast cancer cells
Authors: Ahmad Rehan , Alam Maroof , Rajabi Hasan , Kharbanda Akriti , Kufe Donald
Published: August 2014

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in about 90% of human breast cancers. The present studies have been performed on breast cancer cells that were stably silenced for MUC1 expression or overexpress the oncogenic MUC1-C subunit. We demonstrate that MUC1-C is sufficient for induction of MEK-ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. Importantly, MUC1-C-induced upregulation of ALDH1A1 expression is associated with marked increases in ALDH activity as detected by the aldefluor assay. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK-C/EBPβ-ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells.

Note: This abstract was not presented at the conference.

Citation Format: Rehan Ahmad, Maroof Alam, Hasan Rajabi, Akriti Kharbanda, Donald Kufe. MUC1-C oncoprotein induces aldehyde dehydrogenase expression and activity in breast cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C66.
Abstract IA18: Activities of wild-type and mutant p53
Authors: Vousden Karen H.
Published: August 2014

The p53 tumor suppressor protein restrains malignant progression through a number of mechanisms, and most cancers show loss of the normal functions of p53. In many cancers this is due to mutations in the p53 gene, leading to the expression of mutant p53 proteins that have lost wild type activity and acquired an oncogenic function that can promote metastasis. We have found that mutant p53 can promote the activation of integrin and cell surface receptor trafficking that depends on Rab-coupling protein (RCP) and which results in constitutive activation of signaling though the EGFR, MET and integrin. Simultaneous loss of p53 and p63 recapitulates the phenotype of mutant p53 both in cells and in vivo, and this function of mutant p53 reflects, at least in part, the inhibition of the p53 family member p63 . Interestingly, the tumor-associated point mutations induce conformational instability of the p53 protein, and the misfolding of p53 correlates with loss of wild type and gain of mutant activity. We have found that folding of wild type p53 is promoted by an interaction with the chaperonin CCT. Intriguingly, p53 proteins mutated to prevent the interaction with CCT show conformational instability, and acquire an ability to promote invasion and random motility that is similar to the activity of tumor derived p53 mutants. Our data therefore suggest that both growth suppression and cell invasion may be differentially regulated functions of wild type p53.

Citation Format: Karen H. Vousden. Activities of wild-type and mutant p53. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA18.
Abstract IA26: Regulation of metastasis in breast cancer
Authors: Werb Zena
Published: August 2014

Breast cancer patients with metastatic disease have poor prognoses. A key transcription factor, the loss of GATA3 by mutation or loss of expression correlates with a worse prognosis. Indeed, GATA3 is the third most mutated gene in breast cancer progression. In mouse and human breast cancer cells, GATA3 suppresses metastasis by promoting tumor cell differentiation and by altering the tumor microenvironment. Although GATA3 has many direct targets, one major pathway downstream of GATA3 regulating metastasis is through its induction of miR29 expression. miR29b is enriched in luminal breast cancers, which are high in GATA3. miR29b inhibits metastasis directly by targeting a network of pro-metastatic regulators involved in angiogenesis, collagen remodeling and proteolysis. Loss of miR29b, even in GATA3-expressing cells increases metastasis and promotes a mesenchymal phenotype. That inhibiting metastasis by regulating tumor microenvironmental genes through a GATA3-miR29b axis opens up potentially new therapies for breast cancer. (Supported by Grant R01 CA129523).

Citation Format: Zena Werb. Regulation of metastasis in breast cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA26.
Abstract IA28: Haploinsufficiency in cancer
Authors: Davoli Teresa , Solimini Nicole L. , Pavlova Natalya N. , Xu Qikai , Mengwasser Kristen , Sack Laura M. , Liang Anthony C. , Schlabach Michael R. , Luo Ji , Burrows Anna E. , Anselmo Anthony N. , Li Mamie Z. , Elledge Stephen J.
Published: August 2014

Breast cancer is a collection of diseases with distinct clinical behaviors and underlying genetic causes. We have searched genetically for genes that have cancer relevant phenotypes including genes that alter cellular proliferation, cellular transformation, cell survival, cellular senescence, and genes that are essential for the proliferation of cancer cells. We have approached this by the generation of libraries of shRNAs for loss of function experiments and libraries of ORFs for gain of function experiments. We will discuss these technologies and how they can be applied to the functional dissection of genes important for breast cancer. Using these new technologies we have identified new oncogenes and tumor suppressors. We find that tumor cells selectively delete negative growth regulators and suggest that the deletion of clusters of these genes may drive tumorigenesis by haploinsufficiency. These recurrent deletions also avoid deletion of one copy of many essential genes through haploinsufficiency. We put forward the Cancer Gene Island model that postulates that tumors select for hemizygous loss of islands of gene enriched in negative regulators of proliferation and depleted in essential genes to promote tumor cell proliferation through cumulative haploinsufficiency.

Citation Format: Teresa Davoli, Nicole L. Solimini, Natalya N. Pavlova, Qikai Xu, Kristen Mengwasser, Laura M. Sack, Anthony C. Liang, Michael R. Schlabach, Ji Luo, Anna E. Burrows, Anthony N. Anselmo, Mamie Z. Li, Stephen J. Elledge. Haploinsufficiency in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA28.
Abstract IA7: Translational control of cancer via eIF4E/4E-BPs
Authors: Sonenberg Nahum
Published: August 2014

Control of translation initiation plays an important role in cell growth, proliferation and cancer development and progression. mRNA translation is dysregulated in many cancers via a combination of protein overexpression and defects in the pathways that signal to the translation machinery. In support of the critical function of translational control in cancer is the discovery of mutations in translational components in genetic syndromes associated with cancer susceptibility.

A key regulator of translation initiation is the mRNA 5'-cap-binding protein, eIF4E. eIF4E overexpression engenders transformation of rodent and human cells and causes cancer in mice. eIF4E levels and phosphorylation are elevated in many types of human cancers. Phosphorylation of eIF4E on Ser209 is required for efficient transformation by eIF4E. We generated eIF4E ‘knock-in’ mice in which Ser209 was mutated to alanine. These mice were resistant to prostate cancer induced by PTEN deletion, or cancers induced by other means.

An important downstream component of the PTEN/Akt/mTOR signaling pathway, which is strongly implicated in cancer etiology, is the translation machinery. A well-characterized target of mTOR is 4E-BP1, which binds to eIF4E and inhibits cap-dependent translation. mTOR forms two distinct complexes, mTORC1 and mTORC2. mTORC1 integrates growth factor and nutrient signals to control cell proliferation (increase in cell number) and cell growth (increase in mass). mTORC1 controls these processes by stimulating mRNA translation via phosphorylation of its two major downstream targets: the 4E-binding proteins (4E-BP1, 2 and 3) and the S6 kinases (S6K1 and 2). 4E-BPs impair the assembly of the eIF4F pre-initiation complex by competing with eIF4G for binding to eIF4E, whereas S6Ks phosphorylate a number of targets including ribosomal protein S6 and eIF4B. We showed that the 4E-BPs do not affect cell size, but strongly inhibit cell proliferation, by selectively inhibiting the translation of mRNAs encoding for proliferation-promoting proteins, and proteins involved in cell cycle progression.

The hyperactivation of the mechanistic/mammalian target of rapamycin (mTOR) occurs in the majority of cancers. Therefore, targeted inhibition of mTOR is an attractive anti-cancer strategy. We show that high expression of eIF4E confers increased resistance of cells to the anti-proliferative effects of asTORi. Conversely, depletion of eIF4E levels by RNAi potentiates the anti-neoplastic effects of asTORi in vitro and in vivo. Anti-sense therapy against eIF4E is presently evaluated in clinical trials against cancers. Our data indicate that combining this strategy with targeted mTOR inhibition could have increased therapeutic benefits.

Citation Format: Nahum Sonenberg. Translational control of cancer via eIF4E/4E-BPs. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA7.
Abstract IA8: Novel insights into synthetic lethal interactions in cancer: Common nodes in protein synthesis control and nucleotide metabolism
Authors: Ruggero Davide
Published: August 2014

Our research is centered on understanding the molecular mechanisms by which impairments in accurate control of mRNA translation, cell growth, and overall cellular protein synthesis rates lead to cancer and human disease. We have uncovered that a common denominator of multiple oncogenic pathways (i.e. PI3K-AKT-mTOR, Ras, Myc) is their ability to directly control the core translation machinery of a cell, resulting in intrinsic regulation of the cancer cell gene expression program. Our work delineates how the “cancer translatome” can reprogram gene expression at the post-transcription level, by controlling the translation of specific subsets of mRNAs through unique cis-acting elements, during distinct steps of cancer progression. The immediate impact of these studies has been the design of a new generation of compounds to target the aberrant translation machinery, presently in clinical trials. I will discuss our most recent findings revealing that oncogenic pathways have evolved self-sustaining circuitries that couple two fundamental cellular processes, protein synthesis and metabolism, to sustain the unchecked growth potential of cancer cells. By employing an unbiased metabolomics approach combined with mouse genetics, our findings unveil a critical feed-forward mechanism by which Myc's ability to elevate the protein synthesis capacity of cancer cells directly reprograms nucleotide biosynthesis pathways as a mechanism to sustain tumor growth and survival. I will further discuss how both PI3K-AKT-mTOR and Myc converge on phosphorylation of 4EBP1, a master regulator of protein synthesis control, setting the stage for a unique therapeutic approach to render Myc druggable in the clinic. Together, our findings reveal that targeting either hyperactivation of protein synthesis or nucleotide production represents a novel and unexpected synthetic lethal mechanism for eradicating cancer but not normal cells.

Citation Format: Davide Ruggero. Novel insights into synthetic lethal interactions in cancer: Common nodes in protein synthesis control and nucleotide metabolism. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA8.
Abstract PR01: Genome wide profiling reveals stimulus-specific functions of p53 in human embryonic stem cells
Authors: Jain Abhinav K. , Akdemir Kadir C. , Allton Kendra , Aronow Bruce , Xu Xueping , Cooney Austin J. , Li Wei , Barton Michelle C.
Published: August 2014

p53 is a critical guardian of somatic cells in the face of cellular stress and genomic challenges. Recent work shows that functions of transcription factor p53 go beyond its well-studied roles in tumor suppression. In addition to functions in cell cycle arrest and apoptosis, p53 is a functional barrier to reprogramming adult cells into induced pluripotent (iPS) cells that are phenotypically similar to embryonic stem (ES) cells. How p53 is regulated and functions in stem cells, is poorly understood. Previously we have shown that p53 plays a significant role that depends on its transcriptional activity in early differentiation of human ES cells (hESCs), as induced in vitro by retinoic acid (RA). In order to delineate p53 transcriptional functions during differentiation and how p53 selectively responds to specific signals in hESCs we performed genome wide profiling of p53 chromatin interactions and target gene expression in hESCs in response to early differentiation or DNA damage. The majority of p53 binding sites is unique to each state and defines stimulus-specific p53 response in hESCs. p53 binding regions specific to differentiation significantly overlap with those of core-pluripotency factors OCT4 and NANOG. Differentiation-specific p53 targets are highly expressed, and include many developmental transcription factors, poised by OCT4/NANOG and bivalent histone marks (H3K4me3 and H3K27me3) in pluripotent hESCs. Activation of poised genes during differentiation is associated with elimination of H3K27me3 marks, which is largely dependent on p53. During hESC differentiation, p53 interacts with H3K37me3 specific de-methylases (JMJD3 and UTX) that are enriched at the upstream regions of poised genes resulting in alteration of bivalent histone status and subsequent gene activation. These studies suggest that p53 plays essential roles in regulating epigenetic bivalency in ES cells. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Thus, our findings expand the registry of p53-regulated genes to define p53-mediated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs.

This abstract is also presented as poster A68.

Citation Format: Abhinav K. Jain, Kadir C. Akdemir, Kendra Allton, Bruce Aronow, Xueping Xu, Austin J. Cooney, Wei Li, Michelle C. Barton. Genome wide profiling reveals stimulus-specific functions of p53 in human embryonic stem cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR01.
Abstract A16: Stromal LOX promotes TGFβR2 null breast cancer metastasis
Authors: Pickup Michael W. , Owens Philip , Gorska Agnes , Chytil Anna , Weaver Valerie , Moses Harold
Published: August 2014

To understand how loss of TGF-β signaling in cancer cells promotes tumor progression, we are evaluating modifications to the extracellular matrix in TGF-β type II receptor KO tumors. Tumor cell invasion is a dynamic process which is regulated by many microenvironmental influences such as ECM composition, MMP activity, and exposure of proper integrin binding sites. Crosslinking of collagen fibers increases the overall stiffness of affected tissue providing the mechanical force necessary for tumor cell migration and invasion. Lysyl oxidase is a potent extracellular enzyme that crosslinks collagen and elastin fibers. The study of human breast cancer cell lines shows that highly invasive cells express higher levels of lysyl oxidase and treatment with a lysyl oxidase inhibitor significantly decreases cellular invasion. Recent reports suggest that hypoxia induced metastasis is facilitated by lysyl oxidase crosslinking of the extracellular matrix. TGF-β is another molecule that is known to promote lysyl oxidase expression and will be the focus of our studies.

In this study we utilize transgenic mice which express PyMT under a mammary epithelial specific promoter and have the type II TGF-β receptor conditionally deleted from mammary epithelial cells. Prior to tumor palpation, we see an increase in collagen deposition as well as tissue tensile strength indicating the presence of a higher degree of collagen crosslinking. In PyMT tumors originating from the RII KO mammary glands, increased deposition and maturation of collagen is present in the tumor microenvironment. However, this is not correlated with an increased expression of collagen genes. Subsequent analysis has shown an increased expression and activity of lysyl oxidase present in these TGFβR2KO PyMT tumors. Increased levels of lysyl oxidase are correlated with increased levels of available TGF-β in the RII KO tumors. Upon in vivo inhibition of lysyl oxidase, with beta-aminoproprionitrile, we see a significant decrease in tumor metastasis to the lungs through the inability to escape into the vasculature. Interestingly, these phenotypes are not driven by hypoxia, as shown in previous publications, and instead appear to be driven by TGFβ signaling in the stroma of these tumors. With the source of the TGFβ driving the increase expression of stromal LOX appears to be infiltrating immune cells, MDSCs. This work points to a new paradigm of LOX induction in tumors and presents a new therapeutic target group for LOX inhibition outside of patients presenting with hypoxic tumors.

Citation Format: Michael W. Pickup, Philip Owens, Agnes Gorska, Anna Chytil, Valerie Weaver, Harold Moses. Stromal LOX promotes TGFβR2 null breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A16.
Abstract A23: Cell autonomous and nonautonomous activities of heat shock factor 1 support tumor initiation, progression, and metastasis
Authors: Scherz-Shouval Ruth , Santagata Sandro , Koeva Martina , Whitesell Luke , Lindquist Susan
Published: August 2014

The heat-shock response is a powerful transcriptional program which acts genome-wide, not only to restore the normal protein folding through the induction of heat shock proteins (HSP), but to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by the Heat Shock Factor 1 (HSF1) transcription factor. We have previously shown that HSF1 plays a fundamental role in tumorigenesis, by promoting the survival and malignance of tumor cells, both in tissue culture and in mouse models of cancer [1].

Recently we demonstrated that HSF1 exerts its role by activating a unique transcriptional program in the cancer cells, that is distinct from the one activated during heat shock [2]. In breast cancer and several other types of carcinoma, we found that high HSF1 protein levels and activation of the HSF1-dependent transcriptional program are associated with poor clinical outcome [3]. Here we show that HSF1 is activated not only in the tumor cells, but also in the stromal cells infiltrating the tumor. Examining human patient samples, we find immunohistochemical evidence for activation of HSF1 in the stroma. Using mouse xenograft models and in vitro co-culture we show that HSF1 in the stroma supports tumor cell growth. Finally, expression profiling and analysis of the DNA binding pattern of HSF1 in tumors and in cell culture indicates that stromal HSF1 supports tumorigenesis by activating a unique, stroma-specific transcriptional program. Taken together, our data suggests that HSF1 acts in the cancer cells and in the stroma to activate distinct, yet complimentary transcriptional programs that will facilitate tumor initiation, progression and metastasis.

References

1. Dai, C., et al., Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell, 2007. 130(6): p. 1005-18.

2. Mendillo, M.L., et al., HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers. Cell, 2012. 150(3): p. 549-62.

3. Santagata, S., et al., High levels of nuclear heat-shock factor 1 (HSF1) are associated with poor prognosis in breast cancer. Proc Natl Acad Sci U S A, 2011. 108(45): p. 18378-83.

Citation Format: Ruth Scherz-Shouval, Sandro Santagata, Martina Koeva, Luke Whitesell, Susan Lindquist. Cell autonomous and nonautonomous activities of heat shock factor 1 support tumor initiation, progression, and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A23.
Abstract A28: Role of PDGF signaling during the progression of breast ductal carcinoma in situ to invasive carcinoma
Authors: Strell Carina , Paulsson Janna , Wärnberg Fredrik , Johansson Hemming , Jirström Karin , Hall Per , Östman Arne
Published: August 2014

Breast cancer is one of the most common cancer diseases; in Sweden over 7000 women are diagnosed yearly and 1500 die from the disease. About 20 % of newly diagnosed breast cancers are ductal carcinoma in situ (DCIS), an early stage of breast cancer. The majority of DCIS will never become malignant but 15-20 % will proceed to invasive carcinoma and/or metastasize. Predicative markers for invasiveness are highly needed to understand which tumors will recur and to prevent over treatment.

Tumor characteristics are determined by interactions between malignant cells and the microenvironment. Cancer associated fibroblasts (CAFs) are a major component of the tumor microenvironment and one important regulator of their phenotype are the PDGFs, which signal through the PDGFα and β receptors (PDGFRs). Our previous studies in invasive breast carcinoma have shown that high stromal PDGFβR expression is associated with shorter survival, particularly in pre-menopausal women.

The aim of this study is to investigate the potential prognostic significance of stromal PDGFRs in the transition of breast DCIS to invasive carcinoma.

We found that both PDGFRs are highly expressed in normal breast stroma, whereas DCIS displays loss of periepithelial stromal PDGFαR. In contrast, patients with a high stromal PDGFβR expression in the DCIS lesion, showed a trend for a higher risk of local recurrence or general disease.

These previously un-recognized potentially differential effects of stromal PDGFαR and PDGFβR signaling in breast cancer progression is presently being further explored in animal studies and in vitro experiments.

Citation Format: Carina Strell, Janna Paulsson, Fredrik Wärnberg, Hemming Johansson, Karin Jirström, Per Hall, Arne Östman. Role of PDGF signaling during the progression of breast ductal carcinoma in situ to invasive carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A28.
Abstract A3: Collagen hydroxylases are essential for breast cancer metastasis
Authors: Gilkes Daniele M. , Chaturvedi Pallavi , Wong Carmen , Bajpai Saumendra , Wei Hong , Hubbi Maimon , Wirtz Denis , Semenza Gregg
Published: August 2014

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies to treat cancer patients. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We identify that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers resulting in enhanced cancer cell invasion and metastasis to lymph nodes and lungs. We establish the prognostic significance of collagen hydroxylase expression in human breast cancer biopsies and evaluate the novel use of ethyl 3,4-dihydroxybenzoate, a hydroxylase inhibitor, which decreases tumor fibrosis and metastasis in a mouse model of breast cancer.

Citation Format: Daniele M. Gilkes, Pallavi Chaturvedi, Carmen Wong, Saumendra Bajpai, Hong Wei, Maimon Hubbi, Denis Wirtz, Gregg Semenza. Collagen hydroxylases are essential for breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A3.
Abstract A37: Activation of FGFR1 signaling in mammary tumors leads to STAT3 activation and chemoresistance
Authors: Chuntova Pavlina , Bohrer Laura , Bade Lindsey , Schwertfeger Kathryn
Published: August 2014

Recent studies have shown that the Fibroblast Growth Factor Receptor 1 (FGFR1) gene locus is amplified in approximately 10% of human breast tumors. Breast cancer patients with an FGFR1 amplification have significantly shorter overall survival potentially due to their increased risk of distant metastasis. Using an inducible FGFR1 system (iFGFR1), we have been working to identify the mechanisms involved in FGFR-induced tumorigenesis. Published studies have shown increased survival and proliferation of cells containing the activated iFGFR1. Examining both mouse and human FGF-responsive cell lines, we found that FGFR activation leads to a decrease in apoptosis, both basally and after treatment with chemotherapy agents. Further studies indicated that activation of FGFR signaling leads to indirect activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) both in vitro and in vivo. STAT3 is commonly activated as a result of IL-6 family member signaling. In our in vitro system, iFGFR1 activation leads to production of cytokines such as LIF and IL-6, which in turn signal in an autocrine fashion to activate the STAT3 pathway. Inhibition of FGFR activity in vivo using the small molecule inhibitor PD173034 in combination with the chemotherapeutic doxorubicin resulted in significant reduction of mouse tumor burden. These results provide evidence for the involvement of FGF receptors in the induction of chemoresistance and the potential benefit of combination therapy. Future studies will pursue the mechanism which drives the resistance to drug-induced apoptosis.

Citation Format: Pavlina Chuntova, Laura Bohrer, Lindsey Bade, Kathryn Schwertfeger. Activation of FGFR1 signaling in mammary tumors leads to STAT3 activation and chemoresistance. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A37.
Abstract A40: Cancer cells form in vivo-like tumors in Gelfoam histoculture
Authors: Yano Shuya , Mii Sumiyuki , Tome Yasunori , Hiroshima Yukihiko , Uehara Fuminari , Miwa Shinji , Hoffman Robert M.
Published: August 2014

Tumors in vivo are well known to behave quite differently from cancer cells cultured on plastic or glass plates (monolayer culture). Cell culture on agar (sphere culture) is used for three-dimensional culture, however tumor spheres on agar rarely invade the substrate. It has been previously demonstrated that 3-D Gelfoam histoculture of tumors enables the cancer cells to maintain in vivo properties, in contrast to monolayer or sphere culture. Cancer cell-cycle kinetics were visualized in Gelfoam histoculture using HeLa cells and MKN45 cells expressing fluorescence ubiquitination cell cycle indicator (FUCCI), whereby quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). Gelfoam histoculture of FUCCI-expressing cancer cells results in tumor-like structures with quiescent cancer cells in the center and proliferating cells at the surface, similar to what occurs in vivo as visualized by confocal microscopy. These results have profound implications for cancer biology, since most cellular and molecular studies of cancer cells were performed on plastic monolayer culture where characteristics of the cells may be quite different from the tumor in vivo.

Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Robert M. Hoffman. Cancer cells form in vivo-like tumors in Gelfoam histoculture. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A40.
Abstract A41: Cancer cells invade or divide, not both
Authors: Yano Shuya , Mii Sumiyuki , Tome Yasunori , Hiroshima Yukihiko , Uehara Fuminari , Miwa Shinji , Hoffman Robert M.
Published: August 2014

Invasive cancer cells are a critical target for chemotherapy. However most chemotherapeutic agents in clinical use target dividing cancer cells. Cancer cell-cycle kinetics were visualized in Gelfoam histoculture using a fluorescence ubiquitination cell cycle indicator (FUCCI), whereby quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). Real-time imaging demonstrated that cancer cells in G0/G1 phase are invasive and disseminate in Gelfoam histoculture. In vivo, FUCCI-expressing cancer cells were in G0/G1 phase when they metastasized to the liver. Disseminated cancer cells in the peritoneal cavity were also in G0/G1 phase. Most drugs currently in use target cells in S/G 2/M, which may not be capable of invasion. Color-coded cell-cycle imaging in Gelfoam histoculture should provide a novel platform for the rapid screening of candidate therapeutic agents against quiescent invasive cancer cells.

Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Robert M. Hoffman. Cancer cells invade or divide, not both. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A41.
Abstract A52: Vimentin serves as a signaling scaffold at focal adhesion sites to regulate lung cancer cell adhesion
Authors: Havel Lauren , Marcus Adam I.
Published: August 2014

Vimentin is a dynamic type III intermediate filament that is overexpressed in various cancers including non-small cell lung cancer (NSCLC). It is a classic biomarker for highly invasive tumors and is associated with increased likelihood of metastasis and poor survival. Vimentin filaments are dynamic and localize to focal adhesions, where they assemble and disassemble to promote cell migration. Our data show that vimentin depletion reduces cell adhesion and phosphorylated focal adhesion kinase (pFAK); therefore, we hypothesize that vimentin acts through FAK to regulate cell adhesion and metastasis. We show that vimentin filaments enter pFAK- and pSrc-positive focal adhesion sites, and upon vimentin depletion, pFAK, pSrc and total FAK levels decrease. To explore the mechanism behind this observation, we performed a phospho-proteomic screen for proteins whose phosphorylation is altered upon vimentin loss. One of the identified proteins was a guanine nucleotide exchange factor (GEF) that regulates rac1. We show that vimentin loss leads to GEF mis-localization and downstream rac1 inactivation. Furthermore, inactivation of the GEF itself resulted in aberrant cellular adhesion and loss of pFAK. Based upon this data, we propose that vimentin serves as a signaling scaffold to regulate FAK via a rac1-GEF interaction. This work provides insight into the mechanism by which vimentin affects cell adhesion and potentially NSCLC metastasis.

Citation Format: Lauren Havel, Adam I. Marcus. Vimentin serves as a signaling scaffold at focal adhesion sites to regulate lung cancer cell adhesion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A52.
Abstract A54: TACE-regulated TGF-alpha shedding drives GRB7-dependent invasion in triple-negative breast cancer cell lines
Authors: Calvo Veronica , Giricz Orsi , Kenny Paraic A.
Published: August 2014

The Epidermal Growth Factor Receptor (EGFR) is frequently expressed in triple-negative breast cancer and is a marker of poor prognosis. We have also recently identified an EGFR-associated adaptor protein, GRB7, as being associated with reduced relapse-free survival in triple-negative breast cancer. In this study we have sought to define the mechanisms underlying the contributions of EGFR and GRB7 to triple-negative breast cancer progression.

Because activating mutations in EGFR are very rare events in breast cancer, we screened breast tumor gene expression profiles to examine the distribution of EGFR ligand expression. Of the six known EGFR ligands, TGFalpha was expressed more highly in triple-negative breast tumors than in tumors of other subtypes. TGFalpha is synthesized as a transmembrane precursor requiring TACE/ADAM17-dependent proteolytic release in order to activate its receptor. In this study we show that an inhibitor of this proteolytic release blocks invasion, migration and colony formation by several triple-negative breast cancer cell lines. Each of these effects of the drug was reversed upon expression of a soluble TGFalpha mutant which does not require TACE activity to function, implicating this one metalloproteinase substrate as playing a critical role in these metastasis-related phenotypes. A cyclic peptide inhibitor of GRB7, G718-NATE, also blocked tumor cell invasion and colony formation in 3D culture. Importantly, cell lines overexpressing the soluble TGFalpha mutant remained sensitive to the effects of the GRB7 inhibitor, suggesting that a linear TACE-TGFalpha-EGFR-GRB7 signaling module is involved. We additionally demonstrate that GRB7 overexpression upregulates several pathways with roles in tumor cell proliferation, migration and survival.

Taken together, these data demonstrate that TGFalpha is the key ligand for EGFR in triple-negative breast cancer and that it drives invasion and proliferation via GRB7. This work suggests that triple-negative breast tumors in which this pathway is active might benefit clinically from either EGFR or TACE inhibitors.

Citation Format: Veronica Calvo, Orsi Giricz, Paraic A. Kenny. TACE-regulated TGF-alpha shedding drives GRB7-dependent invasion in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A54.
Abstract A57: Characterization of novel regulators of epithelial-mesenchymal transition in human cancer cells
Authors: Li Linna , Ai Nanping , Hulsurkar Mohit , Li Wenliang
Published: August 2014

The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by cancer cells of several cancer types. Recently, a few kinases, such as FYN and c-Met, were reported to participate in regulating EMT. However, the roles of the majority of >700 human kinases in EMT and cancer progression are still unknown. Here we carried out human kinase cDNA screens to identify novel regulators of EMT. Many unknown EMT regulators and a few known ones such as PI3K, PDGFRA, FYN and c-Met, were found to up-regulate mesenchymal marker vimentin. Some also increase expression of fibronectin and N-cadherin, as well as EMT transcriptional regulators, such as ZEB1/2, Snail and Slug. At the same time, the cells acquired mesenchymal phenotypes, such as increased migration/invasion activity, enhanced capability to survive and proliferate in growth factor deprivation condition or under drug treatments. Interestingly, FACS analysis also showed a substantial increase in CD44hiCD24low population in kinase-transducted mammary gland epithelial cells. The increase of CD44hi population could be attributed to increase of a CD44 mesenchymal variant — the standard form CD44s and decrease of the epithelial variant CD44 v8-10. And this alternation was associated with down-regulation of ESRP1 and ESRP2, two splicing factors that regulate CD44 splicing and EMT. Furthermore, the mammary gland epithelial cells undergoing EMT showed increase capability of forming mammosphere and differentiating into other lineage, such as osteoblast and adipocyte. Importantly, some of these novel EMT regulators are associated with tumor progression and stemness of human cancers. Further study of these novel EMT regulators promises to provide new mechanisms of cancer progression, and may also lead to development of new prognostic markers and therapeutics for metastasis.

Citation Format: Linna Li, Nanping Ai, Mohit Hulsurkar, Wenliang Li. Characterization of novel regulators of epithelial-mesenchymal transition in human cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A57.