bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A58: Loss of Par3 promotes metastasis of ErbB2 transformed mammary epithelial cells by inhibiting E-cadherin adhesion junction maturation
Authors: Xue Bin , Muthuswamy Senthil
Published: August 2014

The mechanisms by which tumor cells metastasize and the role cell polarity proteins play in this process are not well understood. We report that Partitioning defective protein 3 (Par3) is dysregulated in metastasis in human breast cancer, and is associated with higher tumor grade and ErbB2-positive status. Downregulation of Par3 cooperated with ErbB2 to induce cell invasion and metastasis in vivo. Interestingly, the metastatic behavior was not associated with an overt mesenchymal phenotype. However, loss of Par3 inhibited E-cadherin junction maturation, disrupted membrane and actin dynamics at cell-cell junctions and decreased cell-cell cohesion in a Tiam1/Rac-GTP pathway dependent manner. Inhibition of this pathway restores E-cadherin junction maturation and blocked invasive behavior of cells lacking Par3 suggesting that loss of Par3 promotes metastatic behavior of ErbB2-induced tumor epithelial cells by decreasing cell-cell cohesion.

Citation Format: Bin Xue, Senthil Muthuswamy. Loss of Par3 promotes metastasis of ErbB2 transformed mammary epithelial cells by inhibiting E-cadherin adhesion junction maturation. [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 A58.
Abstract A59: Dixdc1 links the LKB1 tumor suppressor to Snail, noncanonical Wnt signaling, and metastasis
Authors: Goodwin Jonathan M. , Svensson Robert U. , Shackelford David B. , Winslow Monte M. , Shaw Reuben J.
Published: August 2014

The kinase LKB1 is a tumor suppressor mutated in familial and sporadic human cancers, whose loss is uniquely coupled with metastatic progression. Although LKB1 controls growth and metabolism through phosphorylation of AMPK, it also phosphorylates the related MARK/ Par1 kinases, which play conserved roles in cell polarity and cytoskeletal dynamics. In a screen for novel targets of LKB1-dependent signals, we identified the scaffold protein DIXDC1 as a substrate of MARK1 which controls cell migration and invasion. DIXDC1 localizes to focal adhesions, and like LKB1, its depletion results in hyperactivation of focal adhesion kinase and subsequent increases in the EMT-transcription factors Snail and Slug. Depletion of LKB1 or DIXDC1 results in Snail-dependent induction of the noncanonical Wnt5a and Wnt5b, which are required for the increased invasive behavior of these cells. Studies on a panel of murine lung adenocarcinoma cell lines from defined stages of metastatic progression reveals that DIXDC1 depletion was sufficient to promote metastasis, while Wnt5a and Wnt5b are required for metastatic behavior in these cells. DIXDC1 is frequently downregulated in human cancers, and inversely correlates with Snail and Wnt5 family members expression. This study reveals the first downstream effector of LKB1 in tumor suppression that is independent of AMPK and identifies a novel signaling pathway that provides a molecular explanation for how LKB1 controls EMT and metastasis.

Citation Format: Jonathan M. Goodwin, Robert U. Svensson, David B. Shackelford, Monte M. Winslow, Reuben J. Shaw. Dixdc1 links the LKB1 tumor suppressor to Snail, noncanonical Wnt signaling, 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 A59.
Abstract A60: Foxf1 transcription factor drives progression of benign lung adenomas to malignant adenocarcinomas
Authors: Wang Xinjian , Balli David , Le Tien , Kalinichenko Vladimir V. , Kalin Tatiana V.
Published: August 2014

Lung cancer is the leading cause of cancer-related deaths in the United States with current 5-year survival only 8—12%. Patients with non-small cell lung cancer (NSCLC) develop metastases prior to clinical symptoms, which reduces successful treatment outcomes. The new therapeutic approaches targeting progression and metastasis of NSCLC are needed. Foxf1 transcription factor is expressed in mesenhyme-derived cells, such as fibroblasts, smooth muscle cells and endothelial cells, but absent from epithelial-derived cells, such as alveolar and airway epithelial cells, and benign lung adenoma cells. In present study, we used immunohistochemistry to demonstrate that Foxf1 expression is up-regulated in the invasive front of human lung adenocarcinomas. Foxf1 expression is also induced in mouse lung adenocarcinomas, caused by either activation of K-Ras G12D transgene or transplantation of Lewis Lung Carcinoma (LLC) cells. In vitro, shRNA-mediated depletion of Foxf1 from LLC cells decreased migration, invasion and colony formation of LLC cells on soft agar. Depletion of Foxf1 reduced mRNA and protein levels of Twist1, and prevented TGFbeta-induced EMT. In vivo, Foxf1-depleted LLC cells failed to form lung metastasis in the transplantation model of lung cancer. Furthermore, we generated transgenic mice with Doxycycline-inducible expression of Foxf1 under control of epithelial-specific SPC-promoter (epFoxf1 mice). Lung adenomas in these mice were induced by urethane. Activation of the Foxf1 transgene in pre-existing lung adenomas promoted their progression towards adenocarcinomas that invaded adjacent bronchiolar lumens and pleural surfaces. Transgenic expression of Foxf1 in tumor cells resulted in the loss of cell polarity, decreased E-cadherin, and increased expression of aSMA, Twist1 and other EMT associated genes. Foxf1 directly bound to promoter region of Twist1, implicating the Foxf1 in the regulation of the Twist1 gene. The present study provides the first genetic evidence that aberrant expression of Foxf1 induces EMT in lung tumors and promotes the progression of lung adenomas to invasive adenocarcinomas, suggesting that Foxf1 is a promising target for anti-cancer therapy.

Citation Format: Xinjian Wang, David Balli, Tien Le, Vladimir V. Kalinichenko, Tatiana V. Kalin. Foxf1 transcription factor drives progression of benign lung adenomas to malignant adenocarcinomas. [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 A60.
Abstract A64: Identification of p75NTR/TrkB-BDNF axis in breast cancer brain metastasis
Authors: Chen Wenhong , McMahan Christine N. , Barlow Keith D. , Juarez Dennis , Metheny-Barlow Linda J.
Published: August 2014

Metastasis of breast cancer to the brain is increasing and is a significant cause of morbidity and mortality for breast cancer patients. In order to understand the molecular mechanisms controlling metastasis to the brain, we have generated two mouse models of breast cancer brain metastasis (4T1.luc2.Br5, Eo771.Br5) via serial selection of these cells through the brains of syngeneic mice following carotid artery injection. In vitro, the 4T1.luc2.Br5 cells exhibit increased proliferation and invasion through basement membrane proteins, while both models demonstrate increased ability to adhere to brain microvascular endothelial cells. Transcriptional profiling followed by Western analysis determined that the p75NTR neurotrophin receptor and its ligand BDNF are highly upregulated in brain-metastatic 4T1.luc2.Br5 cells. The TrkB neurotrophin receptor for BDNF is expressed by these cells as well. Knockdown of p75NTR abolishes the increased invasive capacity of brain-selected 4T1.luc2.Br5 in vitro, confirming a role for p75NTR signaling in invasion. While p75NTR expression in primary breast epithelial cells is reportedly low, we observed that 8 of 12 (67%) human breast cancer brain metastasis specimens express high levels of p75NTR, further suggesting an important role in metastasis to brain. Interestingly, while the brain metastatic Eo771.Br5 cells do not express p75NTR in vitro, both the TrkB receptor and its BDNF ligand are upregulated compared to parentals. Together these data suggest a role for activation of p75NTR/TrkB-BDNF signaling in breast cancer brain metastatic cells, and that targeting this pathway may be a means to prevent or treat breast cancer brain metastasis.

Citation Format: Wenhong Chen, Christine N. McMahan, Keith D. Barlow, Dennis Juarez, Linda J. Metheny-Barlow. Identification of p75NTR/TrkB-BDNF axis in breast cancer brain 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 A64.
Abstract A65: ShcA is required for the TGFβ-induced repression of Chordin-like-1 in ErbB2-expressing breast cancer cells
Authors: Northey Jason J. , Dong Zhifeng , Cyr-Depauw Chanele , Cory Sean , Siegel Peter M.
Published: August 2014

Clinical data and transgenic mouse models have provided clear evidence of a critical role for the adaptor protein ShcA in mammary tumor formation and metastasis downstream of several oncogenes. Indeed, we have demonstrated that stable silencing of ShcA expression in ErbB2-expressing breast cancer cells impairs tumor outgrowth and metastasis in vivo. In addition, we have identified a requirement for an intact PTB domain and the three tyrosine residues in ShcA for mediating TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. Together, these observations position the ShcA adaptor as an important signaling node that facilitates cooperation between the ErbB2 and TGFβ signaling pathways in breast cancer progression. To understand how ShcA signaling contributes to TGFβ-induced gene expression changes, we generated a doxycycline-inducible system to deplete ShcA levels from ErbB2-expressing breast cancer cells. The inducible silencing of ShcA in pre-established ErbB2-positive tumors resulted in impaired tumor cell proliferation and survival in vivo. Gene expressing profiling was then performed on cells with an inducible loss of ShcA expression to identify ShcA-dependent transcriptional responses downstream of active TGFβ and ErbB2 signaling in breast cancer cells. ShcA was found to mediate the suppression of Chordin-like 1, a BMP antagonist. Conditioned media taken from TGFβ-stimulated ErbB2-expressing cells treated with doxycycline to achieve reduced ShcA levels was confirmed to contain increased levels of Chordin-like-1 compared with unstimulated controls. This same conditioned media was efficient at inhibiting the BMP-stimulated phosphorylation of Smad proteins 1/5/8 in ErbB2-expressing cells. Furthermore, this TGFβ upregulation of Chordin-like 1 production resulting from ShcA loss correlated with decreased activation of Smad1/5/8 in ShcA-deficient tumor cells in vivo, and may represent a transcriptional mechanism through which ShcA coordinates ErbB2 and TGFβ initiated tumor growth and invasion.

Citation Format: Jason J. Northey, Zhifeng Dong, Chanele Cyr-Depauw, Sean Cory, Peter M. Siegel. ShcA is required for the TGFβ-induced repression of Chordin-like-1 in ErbB2-expressing breast 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 A65.
Abstract A66: NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling
Authors: Pugacheva Elena N. , Loskutov Yuriy , McLaughlin Sarah , Kozyulina Polina , Kozyreva Varvara , Ice Ryan , Culp Mark , Wysolmerski Robert , Weed Scott , Ivanov Alexey
Published: August 2014

The adhesion scaffolding protein NEDD9 was identified as potential pro-metastatic gene in several cancers. The molecular mechanisms of NEDD9-driven metastasis are still unknown. In this study, we show that expression of NEDD9 positively correlates with the invasive stage of breast cancer. We show that NEDD9 localizes to invadopodia and endosomes. Notably, NEDD9 depleted cells have increased levels of inactive surface receptors due to increase in fast recycling of Rab4 and Rab5 positive vesicles. Mechanistically, we found that NEDD9 binds to and scaffolds the Arf6 specific GAP - ASAP3, decreasing Arf6 activity. Thus, depletion of NEDD9 leads to activation of Arf6. Inhibition of Arf6 or re-expression of NEDD9 in shNEDD9 cells was sufficient to restore recycling rates, decrease the number of Rab4 and 5 positive vesicles and the invasive properties of tumor cells. Thus, in this work, we uncover the mechanistic basis of NEDD9-driven invasion and identify a new role for NEDD9 in Arf6-dependant endocytosis.

Citation Format: Elena N. Pugacheva, Yuriy Loskutov, Sarah McLaughlin, Polina Kozyulina, Varvara Kozyreva, Ryan Ice, Mark Culp, Robert Wysolmerski, Scott Weed, Alexey Ivanov. NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling. [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 A66.
Abstract A69: aB-crystallin: A novel regulator of breast cancer metastasis to the brain
Authors: Malin Dmitry , Strekalova Elena , Ugolkov Andrey , Petrovic Vladimir , Cryns Vincent
Published: August 2014

Brain metastases are a devastating complication of advanced breast cancer that result in disabling neurologic deficits and death within one year in the vast majority of patients. Triple-negative breast tumors, which lack expression of the estrogen receptor, progesterone receptor and HER2, commonly metastasize to the brain. However, the genes regulating brain metastasis are poorly understood. The molecular chaperone aB-crystallin is frequently expressed in triple-negative breast cancer and has been reported to play important roles in cytoskeletal organization, cell migration and invasion. As such, we hypothesized that aB-crystallin promotes metastasis in triple-negative breast cancer. To test this hypothesis, we stably overexpressed aB-crystallin in triple-negative breast cancer cells with low endogenous expression of the protein and stably silenced aB-crystallin in triple-negative breast cancer cells with high endogenous levels. We examined the effects of altering aB-crystallin levels on adhesion to human brain microvascular endothelial cells (HBMECs) and penetration of an in vitro model of the blood-brain barrier (BBB). In addition, orthotopic models of triple-negative breast cancer in which fluorescently labeled breast cancer cells metastasize from the mammary gland to the brain were utilized to evaluate the effects of aB-crystallin on primary and metastatic tumor burden in vivo. We observed that silencing aB-crystallin inhibited adhesion to HBMECs and penetration of an in vitro BBB model, while overexpression of aB-crystallin enhanced adhesion to HBMECs and penetration of the BBB in vitro. Consistent with these results, overexpression of aB-crystallin in triple-negative breast cancer cells increased brain metastasis in female NSG mice with transplanted mammary tumors, while silencing aB-crystallin inhibited brain metastasis. We also demonstrated that aB-crystallin enhanced adhesion to HBMECs by a b1 integrin-dependent mechanism. Taken together, our results point to a novel role of aB-crystallin in breast cancer brain metastasis and suggest that aB-crystallin may be a promising drug target for this rapidly fatal disease.

Citation Format: Dmitry Malin, Elena Strekalova, Andrey Ugolkov, Vladimir Petrovic, Vincent Cryns. aB-crystallin: A novel regulator of breast cancer metastasis to the brain. [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 A69.
Abstract A7: Neural regulation of metastasis: New avenues for therapeutic intervention
Authors: Le Caroline P. , Lamkin Donald , Chai Ming G. , Kim Corina , Pimentel Matthew A. , Cole Steven W. , Sloan Erica K.
Published: August 2014

Recent clinical and experimental studies demonstrated that the neuroendocrine system accelerates cancer development and progression. To investigate the cellular and molecular mechanism of neuroendocrine regulation of cancer, we have used advanced in vivo imaging technologies to investigate spatial relationships between peripheral neural system, tumor cells and the metastatic microenvironment. We found that chronic activation of the sympathetic nervous system accelerates breast cancer metastasis to distant tissues. Increased metastasis was dependent on beta-adrenergic signaling which induced macrophage recruitment to primary tumors and induced a switch to pro-metastatic and pro-inflammatory gene expression. Our recent studies in orthotopic xenograft models of pancreatic cancer and leukemia showed that activation of neural stress-response pathways similarly accelerated disease progression, suggesting that adrenergic signaling may be a general physiological regulator of cancer progression. These findings raise the possibility that beta-blockade of neural stress response pathways may be a novel therapeutic strategy to slow or prevent metastasis. In addition, we will discuss findings that targeting peripheral nerve fibers may provide therapeutic leverage against metastasis.

Citation Format: Caroline P. Le, Donald Lamkin, Ming G. Chai, Corina Kim, Matthew A. Pimentel, Steven W. Cole, Erica K. Sloan. Neural regulation of metastasis: New avenues for therapeutic intervention. [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 A7.
Abstract A73: In vivo evidence of TIMPv as a potential target of CD146-signaling inhibiting breast tumor invasion
Authors: Ouhtit Allal , Elmageed Zakariya Y. Abd , Bhat Sunil , Raman Pravrutha , Al-Jardani Suad H. , Gupta Ishita , Shanmuganathan Somya , Al-Riyami Hamad
Published: August 2014

CD146, also known as melanoma cell adhesion molecule (Mel-CAM, MCAM) or MUC18, was initially identified as a marker of melanoma progression and a promoter of melanoma and prostate cancer (PC) metastasis. In contrast, CD146 appears to play a tumor suppression role in breast cancer (BC). Here, we applied microarray gene expression profiling analysis, using a tetracycline (tet On)-inducible system of CD146 in the MDA-MB-231 BC cell line, and we identified TIMPv (a variant of Tissue Inhibitor of Metalloproteinases), showing a 7 fold increase, as a potential CD146-downstream signaling transcriptional target; this was confirmed in vitro by both time-course western blotting and RT-PCR. Furthermore, breast tumor tissues from both Omani patients and patients from New Orleans area in Louisiana, were examined by immunohistochemistry for the expression of CD146 and TIMPv. Strikingly, the results revealed that the expression levels of both CD146 and its downstream target TIMPv showed parallel inhibition patterns in BC progression, with high expression in normal and benign but low or no expression in malignant and metastatic breast tumor tissue. These findings support the hypothesis that the expression of both, CD146 and its downstream target TIMPv, is lost during BC progression and metastasis. Ongoing studies in our laboratory aim to shed light on the molecular components linking CD146 to the activation of TIMPv transcription.

Citation Format: Allal Ouhtit, Zakariya Y. Abd Elmageed, Sunil Bhat, Pravrutha Raman, Suad H. Al-Jardani, Ishita Gupta, Somya Shanmuganathan, Hamad Al-Riyami. In vivo evidence of TIMPv as a potential target of CD146-signaling inhibiting breast tumor invasion. [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 A73.
Abstract A76: FGF-2 accelerates TGF-β-induced epithelial-mesenchymal transition
Authors: Saitoh Masao , Shirakihara Takuya , Miyazawa Keiji
Published: August 2014

Epithelial mesenchymal transition (EMT) is a crucial event in appropriate embryonic development as well as in wound healing, tissue repair, and cancer progression in adult tissues. EMT endows cells with migratory and invasive properties, inhibits apoptosis and senescence, contributes to immunosuppression, and induces stress resistance and stem cell properties. We have recently demonstrated that transforming growth factor (TGF)-β induces isoform switching of fibroblast growth factor (FGF) receptors through regulating expression of epithelial splicing regulatory proteins 1 and 2, and thereby FGF-2 promotes EMT in collaboration with TGF-β. We performed a microarray analysis to elucidate changes in expression profiles during the enhanced EMT by combined treatment of TGF-β with FGF-2, and found that several genes are specifically upregulated by the combined treatment. Here, roles of these genes in the enhanced EMT by TGF-β and FGF-2 will be discussed.

Citation Format: Masao Saitoh, Takuya Shirakihara, Keiji Miyazawa. FGF-2 accelerates TGF-β-induced epithelial-mesenchymal transition. [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 A76.
Abstract A77: Transmembrane 4 L six family member 5 (TM4SF5) regulates FAK/c-Src signaling activity for cell migration and invasion
Authors: Jung Oisun , Choi Yoon-Ju , Lee Jung Weon
Published: August 2014

Transmembrane 4 L six family member 5 (TM4SF5) is a transmembrane glycoprotein of the transmembrane 4 L six family and highly expressed in many types of cancers. In addition to diverse tumorigenic functions, TM4SF5 also plays roles in migration and invasion, leading to lung metastasis of TM4SF-positive cells injected into tail veins. To understand the TM4SF5-mediated metastatic roles, we mechanistically explored TM4SF5-mediated signaling pathways for migration and invasion. Using endogenously or exogenously TM4SF5-null or -expressing hepatocytes in vitro and mouse animals in vivo, we revealed how TM4SF5 mediated the metastatic processes along the leading edges of migratory/invasive cells. The intracellular loop of TM4SF5 directly and adhesion-dependently bound FAK with causing a structural alteration possibly to release the inhibitory intramolecular interaction, which activated FAK at the leading edges of cells to promote migration/invasion and in vivo metastasis. Impaired interactions between TM4SF5 and FAK attenuated phospho-FAK, signaling link to actin organization machinery, and metastatic potentials. Meanwhile, the cytosolic C-terminus of TM4SF5 bound c-Src that led to invasive protrusion formation. Wildtype TM4SF5 expression enhanced invasive protrusion formation in a c-Src-dependent manner, compared with TM4SF5-null control hepatocytes, but tailless TM4SF5ΔC cells were more efficient than were wildtype TM4SF5 cells, suggesting a negatively regulatory role by its C-terminus. c-Src activity of TM4SF5 WT- or TM4SF5ΔC-expressing cells EGF-independently correlated with enhanced phospho-Tyr845 in EGFR for the invasive protrusions. Altogether, this study suggests that TM4SF5 is involved in signaling for migration and invasion and is a promising target for the treatment of TM4SF5-positively metastatic cancer.

Citation Format: Oisun Jung, Yoon-Ju Choi, Jung Weon Lee. Transmembrane 4 L six family member 5 (TM4SF5) regulates FAK/c-Src signaling activity for cell migration and invasion. [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 A77.
Abstract A78: MUC1 drives c-Met-dependent migration and scattering
Authors: Horm Teresa M. , Bitler Benjamin G. , Broka Derrick M. , Louderbough Jeanne , Schroeder Joyce A.
Published: August 2014

The transmembrane mucin MUC1 is overexpressed in most ductal carcinomas, and its overexpression is frequently associated with metastatic progression. MUC1 can drive tumor initiation and progression via interactions with many oncogenic partners, including β-catenin, the epidermal growth factor receptor (EGFR) and Src. The decoy peptide PMIP (Protein transduction domain MUC1 Inhibitory Peptide) has been shown to inhibit the tumor promoting activities of MUC1 in breast and lung cancer, including cell growth and invasion, and its usage suppresses metastatic progression in mouse models of breast cancer. To further characterize the reduced metastasis observed upon PMIP treatment, we performed motility assays and observed that PMIP inhibits cell motility of breast cancer cells. To determine the mechanism by which PMIP inhibits motility, we evaluated changes in global gene transcription upon PMIP treatment, and identified a number of genes with altered expression in response to PMIP. Among these genes is the metastatic mediator, c-Met, a transmembrane tyrosine kinase that can promote cell scattering, migration and invasion. To further investigate the role of c-Met in MUC1-dependent metastatic events, we evaluated the effects of MUC1 expression and EGFR activation on breast cancer cell scattering, branching and migration. We found that MUC1 strongly promoted all of these events and this effect was further amplified by EGF treatment. Importantly, the effect of MUC1 and EGF on these phenotypes was dependent upon c-Met activity. Overall, these results indicate that PMIP can block the expression of a key metastatic mediator, further advancing its potential use as a clinical therapeutic.

Citation Format: Teresa M. Horm, Benjamin G. Bitler, Derrick M. Broka, Jeanne Louderbough, Joyce A. Schroeder. MUC1 drives c-Met-dependent migration and scattering. [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 A78.
Abstract A79: Elucidating the role of E2F2 transcription factor in mediating human breast cancer metastasis
Authors: Yuwanita Inez , Barnes Danielle , Andrechek Eran
Published: August 2014

In human breast cancer metastasis, it is not the primary tumor that is the cause of mortality but metastasis to distant sites. Therefore, it is important to elucidate the biological mechanism that underlies the metastatic processes. To address this, we utilized bioinformatic methods in combination with biochemical assays and genetic studies. Specifically, we crossed a MMTV-Myc mouse model with various activator E2Fs mutants and found that the tumor incidence decreased in the MMTV-Myc mice crossed with E2F2 and E2F3 mutant background. Surprisingly, we also found that the loss of E2F2 sharply increased lung metastasis in MMTV-Myc mice. Here, we test whether these observations in mice translate to human breast cancer and identify the genes under the control of E2F2 transcription factor that are responsible for mediating breast cancer metastasis. To achieve these aims, we examined the probability of activation of the E2F2 transcription factors in human breast cancer. We calculated the probability of E2F2 activation and used this data to stratify the human breast cancer samples and their associated metastatic clinical data. We performed hierarchical clustering of human and mouse gene expression datasets and uncovered two clusters of human breast tumors in which E2F2 played distinctly different roles in mediating metastasis. The human breast cancer samples that clustered with our mouse gene expression data exhibited the same role for E2F2 whereby it increased time to distant metastasis. We then examined genes that were differentially regulated within this cluster and found that there was a set of genes that were associated with a difference in human metastasis survival times and with the E2F2 knockout in the mouse model. To further explore the mechanism by which E2F2 mediates human breast cancer metastasis, we have identified four genes asputative E2F2 target genes including KLK1, MYH2, PTPRD and TNNC2. We found that partial knockdown of KLK1 promotes the migratory capacity of MCF7 cell in vitro. In addition, partial knockdown of PTPRD affected cellular morphology, where they were found to grow in discreet clusters while having little effect on the migration capacity in a scratch assay. We are currently exploring the effects of MYH2 and TNNC2 ablation on these cells. Taken together, we have begun to elucidate the roles of E2F2 transcription factor in mediating breast cancer metastasis as well as establishing a mechanism by which E2F2 regulates metastasis.

Citation Format: Inez Yuwanita, Danielle Barnes, Eran Andrechek. Elucidating the role of E2F2 transcription factor in mediating human 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 A79.
Abstract A84: Synaptojanin-2 and microRNA-31 control invadopodia and metastasis by regulating vesicular trafficking
Authors: Ben-Chetrit Nir , Chetrit David , Koerner Cindy , Carvalho Silvia , Symons Marc , Schmitt Fernando , Wiemann Stefan , Ehrlich Marcelo , Yarden Yosef
Published: August 2014

Growth factors propel cell migration and metastasis, but underlying mechanisms are incompletely understood. Employing EGF-stimulated mammary cells we link the lipid phosphatase synaptojanin 2 (SYNJ2) to an invasive phenotype, and relate high SYNJ2 and low microRNA-31, a metastasis suppressor that restrains SYNJ2, to short survival rates of cancer patients. Knockdown of SYNJ2 robustly impaired both intravasation and extravasation of mammary tumor cells in an animal model. In vitro, SYNJ2-depleted cells exhibited derailed trafficking of EGFR and integrins, resulting in deformed focal adhesions, arrested lamellipodia and disappearance of invadopodia. We conclude that recycling of active EGFRs focally promotes SYNJ2-mediated dephosphorylation of specific phosphoinositol lipids, thereby instigates formation of both invadopodia and lamellipodia and facilitates tumor progression.

Citation Format: Nir Ben-Chetrit, David Chetrit, Cindy Koerner, Silvia Carvalho, Marc Symons, Fernando Schmitt, Stefan Wiemann, Marcelo Ehrlich, Yosef Yarden. Synaptojanin-2 and microRNA-31 control invadopodia and metastasis by regulating vesicular trafficking. [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 A84.
Abstract A86: SIRT1 regulates Rac1-GTPase activation via the guanine nucleotide exchange factor TIAM1
Authors: Saxena Madhurima , Dykes Samantha S. , Wang Allison E. , Cardelli James A. , Pruitt Kevin
Published: August 2014

SIRT1 deacetylase is overexpressed in different types of cancers and we and a few others have described a role for SIRT1 in regulating cancer cell migration. Rho GTPases are classic regulators of cell motility and are known to aberrantly enhance cancer cell migration in response to several anomalous cues. We hypothesized that one of mechanisms employed by SIRT1 to regulate cell motility is through regulation of Rac1-GTPase activation.

To address this we utilized two cancer cell lines, CFPAC-1 pancreatic and MDA-MB-231 breast cancer cells, as our model. A significant decrease was observed in active Rac1 (GTP-bound Rac1) levels, in a GST-PAK-binding domain pull-down assay, upon inhibition of SIRT1 by small molecule inhibitors or knockdown of SIRT1 by shRNA as compared to vehicle-treated or non-targeting shRNA transfected cells. Subsequent analysis revealed that SIRT1 co-immunoprecipitated with the Rac-guanine nucleotide exchange factor (GEF), TIAM1. To test whether TIAM1 is acetylated and if SIRT1 regulates its deacetylation we performed antibody-based pull-down of acetylated lysines from whole cell protein extracts and observed TIAM1 acetylation. Inhibition of deacetylase activity by small molecule inhibitors or overexpression of acetyltransferases caused an enhancement of acetylated TIAM1 signal. While a change in TIAM1 localization was not observed with SIRT1 knockdown, its ability to activate Rac1 was compromised.

Several studies have demonstrated SIRT1's pro-tumorigenic potential. In this study we elucidate a mechanism by which SIRT1 regulates Rac-GTPase activation via TIAM1 acetylation. Controlling the “switch-mechanism” that the small GTPases utilize for inducing downstream activity can be crucial from a drug-development perspective. Our findings not only provide a new mechanism for regulation of Rac-GTPase activity but also emphasize SIRT1's potential as a targetable molecule in cancer.

This work is supported by CA155223 and the graduate stipend for Madhurima Saxena is provided by a Carroll-Feist Pre-doctoral Fellowship awarded by the Feist-Weiller Cancer Center at LSU Health Shreveport, Shreveport, LA.

Citation Format: Madhurima Saxena, Samantha S. Dykes, Allison E. Wang, James A. Cardelli2,3, Kevin Pruitt1,3. SIRT1 regulates Rac1-GTPase activation via the guanine nucleotide exchange factor TIAM1. [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 A86.
Abstract A99: DCQ is a hypoxia-activated quinoxaline 1,4-dioxide that reduces breast cancer metastasis
Authors: Ghattass Khaled I. , Sitt Sally El , Zibara Kazem , Haddadin Makhlouf MJ , El-Sabban Marwan , Ghali-Muhtasib Hala
Published: August 2014

Although tumor hypoxia is associated with the complex processes of tumor metastasis and resistance, and poses significant challenges against traditional cancer treatments, it provides a target for therapy by bioreductive drugs with enhanced cytotoxic activity in the reduced microenvironment of tumors. We identified a potent synthetic quinoxaline di-N-oxide (DCQ) which preferentially targets hypoxic tumors. Here we show that DCQ: 1) Reduces the viability of breast cancer cell lines (MDA-MB-231, MCF-7), 2) Induces reactive oxygen species (ROS)-dependent p53-independent apoptosis, 3) Inhibits the accumulation of hypoxia inducible factor (HIF-1α) via distinct mechanisms in the two cell lines and 4) Significantly reduces hypoxia-induced invasion of breast cancer cell lines in vitro and in vivo. DCQ-induced ROS was associated with increased DNA damage in MDA-MB-231 and MCF-7 cells. The inhibition of HIF-1α accumulation in MCF-7 was in part via the activation of p53 and was accompanied by a decrease in the levels of phosphorylated mTOR, indicating that DCQ-induced reduction of HIF-1α is possibly occurring at the translational level of the protein. HIF-1α reduction was associated with a decrease in p21 and a reversal of the hypoxia-induced VEGF secretion and invasion, which are key processes in tumor metastasis. In MDA-MB-231 cells; however, DCQ reduced HIF-1α through proteasomal degradation, which was accompanied by a reduction of TWIST, a major player in epithelial to mesenchymal transition (EMT). The inhibitory effect of DCQ on hypoxia induced breast cancer invasion was validated in the xenograft model of subdermally injected MDA-MB-231 cells in immune-compromised mice. DCQ significantly increased the survival rate of treated animals. This increase was associated with a reduction of the metastatic dissemination of breast cancer cells into the liver and lungs of DCQ treated mice.

Citation Format: Khaled I. Ghattass, Sally El Sitt, Kazem Zibara, Makhlouf MJ Haddadin, Marwan El-Sabban, Hala Ghali-Muhtasib. DCQ is a hypoxia-activated quinoxaline 1,4-dioxide that reduces 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 A99.
Abstract B1: Hypoxia inducible factor (HIF) promotes reductive carboxylation by regulating citrate levels and renders VHL-deficient and hypoxic cells sensitive to glutamine deprivation
Authors: Gameiro Paulo , Metelo Ana , Pérez-Carro Rojo , Alexandra Arreola , Wang Zongwei , Rathmell Kimryn W. , Olumi Aria , López-Larrubia Pilar , Stephanopoulos Gregory , Iliopoulos Othon
Published: August 2014

Hypoxia is a hallmark of tumor microenvironment. We showed before that exposure of cells to hypoxia reprograms cell metabolism and directs an IDH1-mediated reductive carboxylation (RC) of glutamine-derived alpha-ketoglutarate (a-KG) for lipogenesis. In addition Von Hippel-Lindau (VHL)-deficient renal cell carcinoma cells use glutamine to generate citrate and lipids through RC of a-KG. To gain insights into the role of HIF and the molecular mechanisms underlying RC we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduces intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate, or knocking-down PDK-1 and ACLY enzymes restored intracellular citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action, to maintain lipogenesis. Expression of HIF renders hypoxic and VHL-deficient cells addicted to glutamine in vitro. VHL-deficient cells displayed significantly less growth than their isogenic counterparts in media containing low glutamine concentration or in the presence of a glutaminase inhibitor. Systemic administration of a glutaminase inhibitor (BPTES) suppressed the growth of VHL-deficient human RCC cell lines as xenografts in nude mice. Lastly, we investigated whether RC occurs in vivo. We metabolically labeled mice bearing VHL-deficient tumors by infusing them with 13C-1-Glutamine for up to 6 hours and we detected the early formation of labeled citrate in the tumors; thus we showed for the first time RC in vivo. Our data provide mechanistic insights into the signaling events that mediate hypoxia-induced RC, strongly suggest that RC is an in vivo phenomenon in growing tumors and highlight potential novel therapeutic approaches for treatment of hypoxic and VHL-deficient tumors based on their metabolic signature.

Citation Format: Paulo Gameiro, Ana Metelo, Rojo Pérez-Carro, Arreola Alexandra, Zongwei Wang, Kimryn W. Rathmell, Aria Olumi, Pilar López-Larrubia, Gregory Stephanopoulos, Othon Iliopoulos. Hypoxia inducible factor (HIF) promotes reductive carboxylation by regulating citrate levels and renders VHL-deficient and hypoxic cells sensitive to glutamine deprivation. [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 B1.
Abstract B103: Proinflammatory signaling by cancer-associated fibroblasts co-evolves during mammary carcinogenesis
Authors: Sharon Yoray , Raz Yael , Alon Lina , Erez Neta
Published: August 2014

Cancer-associated fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. We demonstrated that CAFs also mediate tumor-enhancing inflammation: CAFs isolated from pre-neoplastic and tumor tissue expressed a pro-inflammatory gene signature and functioned to recruit macrophages and enhance tumor angiogenesis in a mouse model of squamous carcinoma. Moreover, we demonstrated that pro-inflammatory signaling by CAFs is operative also in mouse and human breast carcinogenesis. Although breast cancer is one of the major tumor types where CAFs were shown to be tumor promoting, there is no detailed analysis of CAF characteristics and function in correlation with tumor progression. We therefore set out to characterize the dynamic changes in CAFs during progression of mammary carcinogenesis, in a transgenic mouse model of human breast cancer, the MMTV-PyMT mice. We show that normal mammary fibroblasts can be “educated” by mammary carcinoma cells to become activated, pro-inflammatory CAFs. Furthermore, transcriptome profiling of fibroblasts isolated from normal mammary glands, or from mammary glands of transgenic mice at defined tumorigenic stages (hyperplasia, early carcinoma, invasive adenocarcinoma) revealed distinct CAF gene expression signatures that correspond to different tumor stages, with only partial overlap between the stages, suggesting co-evolution of the microenvironment with tumorigenic progression. Interestingly, the gene signature of fibroblasts isolated from hyperplastic lesions is inverse to that in CAFs from neoplastic stages, and has a putative growth inhibitory phenotype, while CAFs from early and late carcinoma express pro-inflammatory and tumor promoting gene signatures. Elucidating the molecular mechanisms by which CAFs mediate cancer-related inflammation in breast cancer will greatly increase our understanding of stromal pathways that contribute to tumorigenesis, in an effort to identify novel stromal molecular targets for combinatorial therapeutic approaches to combat breast cancer progression.

Citation Format: Yoray Sharon, Yael Raz1,2, Lina Alon, Neta Erez. Proinflammatory signaling by cancer-associated fibroblasts co-evolves during mammary carcinogenesis. [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 B103.
Abstract B13: Porphyromonas gingivalis-induced autophagy suppresses cell proliferation through G1 arrest in oral cancer cells
Authors: Cho Tae Jin , Woo Vok Hee , Choi Jeom Il , Kim Seung Jo , Shin Hong In , Park Bong Soo , Park Dae Geun , Park Hae Ryoun
Published: August 2014

Epidemiological studies have shown that chronic inflammation predisposes individuals to different types of cancer and that microbial infections can trigger chronic inflammation. Porphyromonas gingivalis is a major microbial pathogen in periodontitis, which is a chronic inflammatory condition of the oral cavity and one of the most common infectious diseases. In this study, we examined the effect of P. gingivalis on oral cancer cells to define the role of chronic periodontitis on the development and/or progression of oral cancers. P. gingivalis-infected cells showed reduced proliferation rates and a concomitant G1 arrest compared with non-infected cells. One mechanism through which P. gingivalis blocked cell cycle progression in oral cancer cells was through ROS production, which induced autophagy. We conclude that the suppression of cell proliferation and cell cycle progression through autophagy may serve as an alternative to apoptosis to eliminate transformed cells. Autophagy represents a survival strategy against environmentally stressful conditions, such as the pathogenic invasion of P. gingivalis, and the prolonged survival of P. gingivalis-infected cells may contribute to the malignant progression of oral cancer cells.

Citation Format: Tae Jin Cho, Vok Hee Woo, Jeom Il Choi, Seung Jo Kim, Hong In Shin, Bong Soo Park, Dae Geun Park, Hae Ryoun Park. Porphyromonas gingivalis-induced autophagy suppresses cell proliferation through G1 arrest in oral 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 B13.
Abstract B15: Invasive breast cancer amplifies lymphangiogenic signals in tumor microenvironment through integrin beta4 expressing cancer-associated fibroblasts
Authors: Kim Baek Gil , Choi Yoon Pyo , Kang Suki , Lee Joo Hyun , Cho Nam Hoon
Published: August 2014

Lymphangiogensis is widely acknowledged as a major route of breast cancer metastasis and a critical alteration of tumor microenvironment (TME) for tumor progression. But, most of it has remained still unknown. Previously, we found that invasive ductal carcinoma (IDC), not ductal carcinoma in situ (DCIS), upregulates integrin alpha6beta4 in the fibrosis adjacent to tumor burden, and that MDA-MB-231 induces integrin beta4 neoexpression in cancer-associated fibroblasts (CAFs) via cell-to-cell contract. Integrin (alpha6)beta4 overexpression is often detected in malignant tumors and negatively associated with patient survivor. In addition, integrin alpha6beta4 was reported to regulate eIF-4E activity leading to vascular endothelial growth factor (VEGF) translation in cancer cells. It is, therefore, plausible that invasive breast cancer cells increase lymphangiogenic signals in TME through driving integrin beta4 expression in CAFs. To verify our hypothesis, we induced integrin beta4 overexpression in CAFs using co-culture with MDA-MB-231 or transfection with pRK5_beta4 plasmid, and then observed tube formation of human dermal lymphatic endothelial cells (HDLEC) by integrin beta4-expressing CAFs in 3D co-culture model. VEGF-D, known as a major factor of lymphangiogenesis, was highly upregulated in integrin beta4-expressing CAFs, which promoted tube formation of HDLEC compared to normal CAFs and MDA-MB-231. Interestingly, the level of VEGF-D expression in MDA-MB-231 was much lower than that of integrin beta4-expressing CAFs before and after co-culture. Taken together, invasive breast cancer may amplify lymphangiogenic signals in TME by inducing integrin beta4-expressing CAFs, and integrin beta4 neoexpression in CAFs by breast cancer may be a pivotal process for lymphangiogensis.

Citation Format: Baek Gil Kim, Yoon Pyo Choi, Suki Kang, Joo Hyun Lee, Nam Hoon Cho. Invasive breast cancer amplifies lymphangiogenic signals in tumor microenvironment through integrin beta4 expressing cancer-associated fibroblasts. [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 B15.