Non-metastatic Line Research Articles

Abstract PS19-25: Metastatic TNBC is highly associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism programmation

Abstract Background: Triple negative breast cancer (TNBC) is characterized by heterogeneous metastatic and metabolic properties, which difficult the diagnosis and development of specific treatment. Therefore, the understanding of the mitochondria´s role in the different grades of TNBC could be relevant for the design of novel specific TNBC treatments. This research aimed to explore the mitochondria morphology and gene expression of proteins related to oxidative and non-oxidative metabolism in metastatic and non-metastatic TNBC cell lines. Methods: We performed a gene expression analysis of mitochondrial biogenesis, EMT and principal metabolism-related genes (Integrated DNA Technologies, CA, USA) in three breast cancer cell lines HCC-1395, MDA-MB-231 and MCF-12A. After the gene expression qPCR data mining, in sílico analisis were performed using confocal microscopy (Leica Microsystems,WZL, DE) in the cell lines where mitochondrial distribution, morphology, function and ROS production were measured. Results: The metastatic TNBC-cell line showed a preference for fatty acid biosynthesis and glycolytic metabolism since overexpression of genes related to both pathways was observed. These metabolism features were accompanied by a fused mitochondrial morphology and lower mitochondrial activity since was observed less mitochondrial density accompanied by the downregulated expression of biogenesis-related genes such as PGC1α. In contrast, the non-metastatic TNBC-cell line presented a hyperfragmented mitochondria, a stress associated mitochondrial morphology accompanied by upregulated expression of mitochondrial biogenesis-related genes, both characteristics related to the higher ROS production observed in this cell line. Conclusions: Metastatic TNBC was characterized by a mitochondrial function and morphology similar to control ranges with a metabolic gene program directed to glycolysis and FA usage. While mitochondria of the non-metastatic TNBC cell line was characterized by a higher density and potential, related to an increase of mitochondrial biogenesis, which in turn was associated with higher levels of ROS and with the consequence of a hyperfragmented mitochondrial morphology. These characteristics can provide a better understanding of the metastasis observed in TNBC and contribute to the development of a specific and personalized TNBC therapy. Key words: Triple Negative Breast Cancer, Mitochondria, Cell-metabolism. Citation Format: Claudia D Aguayo-Millán, Perla Perez-Trevino, Sandra Santuario-Facio, Alberto Camacho, Noemí Garcia, Rocio Ortiz-Lopez. Metastatic TNBC is highly associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism programmation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-25.

Abstract 2907: Exosome secretion is an inheritable property of cancer cells: Single-cell profiling of exosome secretion

Abstract Decades of research on exosomes, nano-sized vesicles secreted by cells, have revealed novel roles of these vesicles in the formation of pre-metastasis niches that enhances the migration of tumor cells to those sites. Paradoxically, more recent work has suggested that these tumor-derived exosomes can also have an immunostimulatory role, depending on the model studied. The rate of secretion of exosomes by single cells is likely heterogeneous, and a deep profiling of the secretion capacity of individual tumor cells has been largely unexplored. We have developed a high-throughput single-cell methodology to quantify the dynamic secretion of exosomes from single cells using a modified immunoassay and sought to define: (a) heterogeneity among individual cells within the tumor cell population, and (b) the nature of the cells secreting exosomes within a tumor cell population. In order to investigate these, we chose to study models of triple negative breast cancer: the metastatic tumor lines, 4T1 (mouse) and MDA-MB-231(human); and the non-metastatic lines 67NR (mouse) and MCF7 (human). Our method revealed that MDA-MB-231 single cells secreted exosomes at a rate, ~2 fold higher than MCF7 cells. Surprisingly, the non-metastatic 67NR cells showed a higher secretion rate (~1.5 fold) than metastatic 4T1 cells. Next, we performed single-cell RNA-seq on 67NR and 4T1 single cells. Consistent with our single-cell exosomal profiling results, 67NR cells were significantly (p-value < 0.01) enriched for genes correlated to exosome formation in comparison to the 4T1 cells. Next, in order to study exosomes of single cells, we isolated single cells using an automated micromanipulator which allowed us to establish monoclonal cell lines. Measurement of these monoclonal cells showed that secretor cells of 67NR secrete exosomes with ~2 fold higher rate than non-secretor cells. Since our in vitro results clearly demonstrated that the secretor clonal cell lines had a higher frequency of exosome secreting single cells, we sought to define the in vivo relevance of these results. Consistent with the hypothesis that the exosomes from 67NR are immune-stimulatory, injection of the secretor clones into Balb/c mice led to lack of primary tumor formation (2/15 mice had tumors) whereas the injection of the non-secretor clones led to tumor formation in 7/15 mice. In aggregate, our results show that the higher rate of secretion of exosomes from non-metastatic cells can facilitate tumor rejection in vivo. We are currently performing in vitro studies with the 67NR and MDA-MB-231 exosomes to study their impact on immune cells. Citation Format: Mohsen Fathi, Robiya Joseph, Melisa Martinez-Paniagua, Jay R Adolacion, Xingyue An, Ankit Mahendra, Konrad Gabrusiewicz, Sujash Chatterjee, Sendurai A. Mani, Navin Varadarajan. Exosome secretion is an inheritable property of cancer cells: Single-cell profiling of exosome secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2907.

Abstract P2-08-04: HMGA1 negatively regulates the expression of PRRX1 in triple negative breast cancer cells, which favors the progression to metastasis

Abstract Background: Triple negative breast cancer (TNBC) presents a very aggressive behavior with a high rate of metastasis. Overexpression of HMGA1 has been reported in TNBC and has been associated with the induction of the Epithelial-Mesenchymal transition (EMT) and metastasis. Therefore, HMGA1 is considered a master regulator of tumor progression in TNBC. The objective of this work was to know which genes are directly or indirectly regulated by HMGA1 to better understand their participation in EMT and their role in aggressive TNBC. Methods: We performed the silencing of the HMGA1 gene using siRNA Silencer® Select Pre-designed (s6667 HMGA1, 4390849 GAPDH, 4390843 Negative control, all from Thermo Fisher, MA, USA) in two TNBC cell lines, HCC-1395 and MDA-MB-231, and we observed the effect of this gene inhibition by microarray global expression analysis using GeneChip Human Genome U133 Plus 2.0 (Affymetrix, CA, USA), comparing the conditions of inhibition versus their own control without inhibition. After the microarray data mining, results for the HMGA1 and PRRX1 genes were validated by qPCR using the Prime Time® Primers for HMGA1 (Hs.PT.58.38699366), PRRX1 (Hs.PT.58.2820749), and GAPDH as endogenous gene (Hs.PT.39a.22214836) with SybrGreen reagent (Roche, Basel, Switzerland). The level of expression of the HMGA1 and PRRX1 proteins was analyzed by Western blot in nuclear protein extracts of each cell line before and after gene silencing. Finally, we performed an in silico analysis using the "Gene 2 promoter" tool in the Genomatix platform to search the promoters and binding proteins of the PRRX1 gene. Results: The silencing of HMGA1 in a non-metastatic TNBC-cell line, HCC-1395, showed deregulation of genes associated with cell proliferation and angiogenesis. Meanwhile the silencing in a TNBC-metastatic cell line, MDA-MB-231, resulted in the deregulation of genes involved in the formation and organization of the cytoskeleton, including the overexpression of PRRX1. Validation of the expression changes of HMGA1 and PRRX1 by qPCR and Western blot was performed and HMGA1 was confirmed to negatively regulate the PRRX1 gene expression. Through in silico studies, we identified several binding sites of HMGA1 to the PRRX1 promoter. Conclusions: The subexpression of PRRX1 is necessary for EMT to occur. In this study, we present the interesting finding that HMGA1 regulates the subexpression of PRRX1, as supported by the experiments of transcriptional and translational expression presented in this work. To our knowledge, this is the first report describing a regulatory role of HMGA1 on PRRX1, which could explain the metastatic capacity of cancers that overexpress HMGA1. Key words: Epithelial-Mesenchymal Transition, Triple Negative Breast Cancer, Mesenchymal-Epithelial Transition, Metastasis. Citation Format: Aguayo-Millán CD, Santuario-Facio SK, Treviño-Alvarado V, Calvo-Anguiano G, Rojas-Martínez A, Camacho-Morales A, Ortíz-López R. HMGA1 negatively regulates the expression of PRRX1 in triple negative breast cancer cells, which favors the progression to metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-04.

Abstract 3264: Genome-wide shRNA screen identifies ITIH5 gene as a metastasis suppressor of pancreatic ductal adenocarcinoma (PDAC)

Abstract Background: PDAC is a leading cause of cancer-related death worldwide, and most patients have incurable disease at diagnosis. Life expectancy is only 3-6 months post-diagnosis, ostensibly due to early dissemination to liver and other vital organs. Results: By transfecting a shRNA library into non-metastatic PDAC line S2-028 followed by experimental metastasis assay, ITIH5 was identified. Metastatic PDAC cell lines (e.g., S2-007, MIAPaCa-2, Panc-1) had significantly lower ITIH5 protein expression compared to immortalized pancreatic ductal epithelial cells and non-/poorly-metastatic PDAC cell lines (e.g., S2-028 and BxPC3). By manipulating expression in different PDAC cell lines (over-expression in S2-007 and MIAPaCa-2, knockdown in non-metastatic S2-028 with two different shRNA) functional and selective regulation of metastasis was observed. Orthotopic tumor growth of PDAC cells was not blocked. Thus ITIH5 qualifies as a metastasis suppressor. Overexpression of ITIH5 significantly inhibited invasion and migration, while down-regulation increased these cellular properties. Immunohistochemistry of a human PDAC tissue microarray (n = 81) revealed that negative expression of ITIH5 showed significantly higher incidence of liver metastasis (P = 0.0482) corresponding with worse prognosis after surgery (P = 0.0299). Conclusions: ITIH5 is a potent metastasis suppressor with suggestive prognostic value in PDAC. Future studies will continue to characterize biochemical properties of ITIH5, identify interaction partners, and investigate the molecular mechanism(s) of action. Support: National Foundation for Cancer Research, Steiner Family Fund and CA168524. Citation Format: Ken Sasaki, Hiroshi Kurahara, S Natsugoe, Tomoo Iwakuma, Danny R. Welch. Genome-wide shRNA screen identifies ITIH5 gene as a metastasis suppressor of pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3264. doi:10.1158/1538-7445.AM2015-3264

Abstract P1-07-20: Melatonin's inhibitory effect on breast cancer metastasis mediated by ROCK-1

Abstract The metastasis occurrence, an important prognostic factor, depends on peculiarities such as cellular invasiveness and cell migration, mechanisms controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). An increased expression of this protein promotes tumor growth and metastasis, a mechanism which can be restricted by the use of the effector's inhibitors. Melatonin, a hormone secreted by the pineal gland, has shown oncostatic action and anti-metastatic effects and can modulate the ROCK-1expression. The objective of this study was to investigate the anti-metastatic response mediated by ROCK-1 and through the action of melatonin and its specific inhibitor (Y27632) in vitro and in vivobreast cancer models. Cells from metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer lines were treated with melatonin and Y27632. Cell viability was verified by MTT assay, cell migration/invasion assays in Boyden chamber, ROCK-1 protein and gene expression by western blot and quantitative real time PCR, respectively. In addition, the in vivometastasis study was performed using female athymic nude mice induced by injection of 2x105 MDA-MB-231 viable cells by tail veinfor lung metastasis and byintracardiac for bone metastasis, during 3 weeks. The animals were treated with melatonin and Y27632 for 2 weeks. The metastasis developments were evaluated by single photon emission computed tomography (SPECT). Treatment with melatonin reduced cell viability and migration of both cell lines (p<0.05). The use of melatonin and Y27632, in association or not, decreased the ROCK-1 protein expression in metastatic cells, not significantly altering its expression in the non-metastatic line (p>0.05). An statistically significant reduction of ROCK-1 gene expression was observed in all treatment groups (p<0.05). ROCK-1 downexpression was more efficient in the group with associated treatments for both lines (p < 0.05). In vivoSPECT images showed multiple foci in the lungs (on Tc-99-tetrofomin images) and in the vertebrae (on Tc-99-MDP images). The numbers of "hot" spots were significantly higher in lung metastasis of control animals compared to treated groups. Semi quantitative analysis showed significantly lower activity in animal lungs that received treatment (p<0.05). The bone metastasis did not show difference between control and treatment groups. Melatonin and Y27632 are effective drugs of metastatic breast cancerin vitro treatment, confirming their effects in decreasing cell viability, invasion, migration and protein expression of ROCK-1 in these cells. In vivo, melatonin and Y27632 treatments seem to be effective reducing lung metastasis but not bone metastasis. Melatonin, in particular, appears to be more effective when combined to ROCK-1 inhibitor. Support: FAPESP. Citation Format: Debora C Zuccari, Thaiz F Borin, Ali S Arbab, Lívia C Ferreira, Marina G Moschetta, Gustavo R Martins, Larissa B Maschio, Vanessa A Fabri, Verena B Coimbra. Melatonin's inhibitory effect on breast cancer metastasis mediated by ROCK-1 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-20.

The Chemical Potential of Cholesterol Regulates the Pro-Metastatic Phenotype in a Cell Culture Model of Breast Cancer

We have developed a simple and reliable method to determine the chemical potential of cholesterol in plasma membranes of living cells in vitro. For a variety of cultured cells, including non-metastatic breast cancer cells, the chemical potential of cholesterol is maintained at a level of about −1.9 kBT per moleculerelative to crystalline cholesterol. But for a metastatic breast cancer cell line, this chemical potential is appreciably greater, at about −0.7 kBT per molecule. In light of these observations and recent reports of cholesterol's role in breast cancer progression we have developed a method to alter and maintain the chemical potential of cholesterol in plasma membranes of cultured cells at any desired level. Through this technique, we lowered the chemical potential of cholesterol in the metastatic cell line to that of the non-metastatic line and found that this significantly reduced the expression levels of several key proteins implicated in breast cancer progression and metastatic spread.

Abstract 3164: Cancer cell adhesion and degradome interact to metastasize

Abstract Background: Adhesion molecules of cancer cells interact with the cancer cell degradome. This is evident from (i) co-localized expression of adhesion molecules such as integrins and degradative enzymes such matrix metalloproteases (MMPs), (ii) regulation of degradome activity by integrin signaling, (iii) key integrins and MMPs binding to the same extracellular matrix (ECM) components, and (iv) interaction between integrins and membrane-bound MMPs in cell-ECM communication in several types of cancer (1-8). Integrins and the degradome have both been implicated in breast cancer metastasis. We are testing for the first time the hypothesis that it is the interactome between these two molecular networks that is critical for breast tumor metastasis to occur. Experimental Design: We performed comparative studies with a panel of non-metastatic (BT-474, T47D, MCF7, SKBr3) versus metastatic (MDA-MB-231, SUM149, SUM159, MDA-MB-468) human breast cancer cell lines and xenografts. All cell lines stably expressed tdTomato fluorescent protein for optical tracking. Comparative immunoblotting, zymography, migration and invasion assays were performed. Enzymatically activatable optical imaging probes IntegriSense (integrins), MMPSense (MMP), and Angiosense (blood vessels) from PerkinElmer were used in our in vivo imaging studies. Primary tumor sizes of 200 mm3 and 600 mm3 were used to account for different stages of tumors growth. Results: Analysis of 1,144 genes from a panel of 28 human breast cancer cell lines from publicly available data bases (9) showed that metastatic breast cancer cells have significantly (p<0.01) higher levels of cell adhesion (integrin α-1,4,5,6,v, integrin β-1, CD44) and ECM degrading enzymes (MMP-2,3,11,14,16,19). Metastatic human breast cancer cell lines expressed elevated protein levels of MMP-1 and MMP-14 compared to nonmetastatic lines. Zymography demonstrated that metastatic breast cancer cells secreted increased amounts of active MMP-2 and MMP-9 compared to non-metastatic cell lines. Metastatic tumor xenografts displayed significantly higher combined MMP-1,-2,-3,-9,-13 activity along with increased integrin expression compared to non-metastatic tumors in vivo. Conclusion: Our results show that elevated activity and expression levels of integrins and MMPs coincide in metastatic breast cancer cell lines and xenografts, which indicates that they may cooperate and interact to enable cancer cell escape from the primary tumor and metastasis. We will perform additional studies to further test our hypothesis by combining cancer cell and tumor studies.

Abstract A11: Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis

Abstract Innate immune cells such as macrophages, monocytes and neutrophils can be recruited to tumors by chemokines, which act on chemokines receptors on these cells. Macrophages and monocytes have well-established roles in promotion of metastasis. However, neutrophils have been reported to have both pro- and anti-metastatic effects. In this study, we used the metastatic 4T1 and non-metastatic 4T07 murine breast carcinoma cells lines (originating from the same tumor) to study effects of neutrophils in breast metastasis. Primary tumors from 4T1 cells, orthotopically injected into syngeneic mice, had more infiltrating neutrophils than did primary tumors from 4T07 cells. The increased numbers of neutrophils, which expressed the chemokine receptor CXCR2, was associated with significantly higher levels of cancer cell-secreted CXCL1, a chemokine ligand for CXCR2. To test whether cancer cell-secreted CXCL1 acted on neutrophils to regulate metastasis, we used shRNAs to reduce CXCL1 expression in the 4T1 cells. Consistent with the lack of CXCR2 expression by the cancer cells, this did not influence cancer cell proliferation or migration in vitro. However, in vivo, tumor infiltration of neutrophils and lung metastasis were both reduced. Moreover, when parental 4T1 cells were orthotopically injected into syngeneic Cxcr2-/-mice, neutrophil infiltration and lung metastasis were significantly decreased as compared to tumors in wild type littermate. These results suggest that paracrine CXCL1 by cancer cells facilitates neutrophil infiltration via CXCR2 and that neutrophils in turn promote lung metastasis. To examine the mechanisms by which interactions between cancer cells and neutrophils might promote lung metastasis, we performed co-culture experiments with cancer cells and neutrophils. We found that neutrophils directly stimulated 4T1 cell invasion through matrigel. Interestingly, cancer-released CXCL1 also promoted the formation of neutrophil extraceullar traps (NETs), which is an expulsion of DNA and proteases from the neutrophils. Both invasion and NET formation were blocked by inhibition of phagocyte NADPH oxidase. Moreover, when mice with 4T1 tumors were treated with a phagocyte NADPH oxidase inhibitor, tumor growth and lung metastasis was reduced. Finally, decreased levels of the phosphorylated form of p47phox, a cytosolic component of phagocyte NAPDH oxidase that is activated by phosphorylation, was decreased in the tumor lysates from 4T1 injected Cxcr2-/- mice as compared to Cxcr2+/- mice. In conclusion, our study revealed that cancer cell-secreted CXCL1 regulates neutrophil infiltration and extracellular trap formation, and that phagocyte NADPH oxidase activity in these neutrophils promoted cancer cell invasion and metastasis. Citation Format: Juwon Park, Jing Qiu, Elizabeth S. Nakasone, Mikala Egeblad. Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support 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 A11.

The Association of Leishmania RNA Viruses with metastatic or mucocutaneous Leishmaniasis in South America

Leishmaniasis is a spectral disease caused by the Trypanosomatid protozoan parasite Leishmania. One manifestation is a disfiguring mucocutaneous form (MCL) involving dissemination to nasopharyngeal areas and tissue destruction, caused by species from the South American Leishmania subgenus Viannia. We reported previously that metastatic L. guyanensis bear high levels of a novel dsRNA totivirus (LRV1), while non‐metastatic lines have low levels or lack LRV1. LRV1‐infected Leishmania were associated with a hyper‐inflammatory response dependent on the host Toll‐like receptor 3 (TLR3) signaling. Thus, LRV in metastatic parasites subverts the host immune response and promotes dissemination. This is the first parasite factor implicated causally in phenotypes relevant to metastatic mucocutaneous disease, with LRV acting as a ‘parasite within a parasite’. The closely related species L. braziliensis is responsible for the great majority of human MCL. Thus we are surveying L. braziliensis strains to establish the distribution and potential for clinical association(s) of LRVs in parasites taken from humans. Several novel LRVs were identified; these are currently being characterized by virus and genome sequencing, classical virological approaches, and infectivity tests of the LRV‐bearing L. braziliensis in animal models.

Abstract 4983: Integrative analysis of DNA copy number and gene expression identifies G3BP1 and HIRA as potential therapeutic targets against metastatic oral squamous cell carcinoma

Abstract The presence of lymph node metastasis is associated with a 50% reduction of 5-year survival in patients with oral squamous cell carcinoma (OSCC). We set out to combine DNA copy number aberrations (CNAs) with gene expression profiles, to identify CNAs-associated genes dysregulated in metastatic OSCC, and determine whether these genes can be targeted to selectively kill metastatic OSCC tumor cells. Toward this end, we interrogated DNA and RNA of the same OSCC cell populations laser micro-dissected from non-metastatic primary tumors (n=17) and metastatic lymph nodes (n=20) using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR) < 5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Out of these, 114 transcripts showed significant correlation between their DNA copy number alternations (estimated using SNPs within 250 kb upstream and downstream of the transcript) and gene expression (FDR < 0.01). Among these CNA-correlated transcripts, 95 had significantly different DNA copy numbers between metastatic and non-metastatic OSCC (FDR <0.01 by Wilcoxon rank test). These 95 transcripts (representing 87 genes) mainly clustered around three genomic locations: 3p25.3-22.1, 9p24.1- 22.3 and 18q21.1-22.3. Among these 87 genes, we selected 28 of the 58 genes that were over-amplified and over-expressed in metastatic OSCC and conducted a high-throughput siRNA-mediated gene knockdown screen in five cell lines derived from primary and lymph node metastatic OSCC. The expression-knockdown of 18 genes showed 30% or more growth suppression in at least one cell line as compared to mock controls in which the cells were treated with transfection reagent only without the siRNA (P < 0.01). In particular, knocking-down G3BP1 and HIRA selectively suppressed the growth of all OSCC cell lines derived from lymph node metastases when compared to non-metastatic lines. Further investigation is warranted to confirm these findings and to examine the biological role of G3BP1 and HIRA in OSCC metastasis and their potential as therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2011-4983

Integrity of SOS1/EPS8/ABI1 Tri-Complex Determines Ovarian Cancer Metastasis

Ovarian cancer is mainly confined in peritoneal cavity and its metastasis is often associated with the formation of malignant ascites. As lysophosphatidic acid (LPA) is present at high levels in ascites of ovarian cancer patients and potently stimulates cell migration, we reason that LPA-stimulated cell migration may play an important role in ovarian cancer metastasis. Here, we show that only ovarian cancer cell lines with LPA migratory response undergo peritoneal metastatic colonization. LPA-stimulated cell migration is required for metastatic colonization because knockdown of LPA receptor subtype 1 (LPAR(1)) abolishes this event. However, the difference in metastatic potentials is not caused by the absence of LPAR(1) because both metastatic and nonmetastatic lines express similar levels of LPAR(1). Instead, we find that LPA can activate Rac only in metastatic cells and that metastatic colonization of ovarian cancer cells necessitates Rac activity. These results thus suggest that LPA-induced Rac activation is a prerequisite for ovarian cancer metastasis. In metastatic cells, Rac activation is facilitated by SOS1/EPS8/ABI1 tri-complex and the integrity of this tri-complex is essential for LPA-stimulated cell migration and metastatic colonization. We show that at least 1 member of SOS1/EPS8/ABI1 tri-complex is absent in nonmetastatic ovarian cancer cells and reexpressing the missing one conferred them with metastatic capability. Importantly, coexpression of SOS1, EPS8, and ABI1, but not of any individual member of SOS1/EPS8/ABI1 tri-complex, correlates with advanced stages and shorter survival of ovarian cancer patients. Our study implicates that the integrity of SOS1/EPS8/ABI1 tri-complex is a determinant of ovarian cancer metastasis.

Metabolomic Changes Accompanying Transformation and Acquisition of Metastatic Potential in a Syngeneic Mouse Mammary Tumor Model

Breast cancer is the most common cancer type for women in the western world. Despite decades of research, the molecular processes associated with breast cancer progression are still inadequately defined. Here, we focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithelial cells to an isogenic series of mammary tumor cell lines with increasing metastatic potentials. Our results suggest a two-step metabolic progression hypothesis during the acquisition of tumorigenic and metastatic abilities. Metabolite changes accompanying tumor progression are identified in the intracellular and secreted forms in several pathways, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, fatty acid and nucleotide biosynthesis, and the GSH-dependent antioxidative pathway. These results suggest possible biomarkers of breast cancer progression as well as opportunities of interrupting tumor progression through the targeting of metabolic pathways.

The Role of microRNA Genes in Breast Cancer Progression and Metastasis.

Abstract MicroRNAs influence tumor progression and metastasis via action as metastasis promoting genes (for example mir-373 and mir-520) or metastasis suppressor genes (for example mir-335). To identify additional microRNAs capable of driving breast cancer metastasis in vivo, we employed a functional genomics screen using the '4T1' syngeneic (Balb/c) mouse model of breast cancer progression. Approximately 400 microRNA sequences were subcloned into the pMSCV_puro retroviral expression vector and the resultant microRNA expression library transduced into fluorescent non-metastatic 67NR-pMSCV_mCherry and mildy-metastatic 66cl4-pMSCV_mCherry cells and selected with puromycin. The transduced cell lines were then implanted into the mammary fat pads of 20 syngeneic Balb/c mice per cell line. Following establishment of primary tumors, the primary tumors and secondary organs (lung, liver, spine, femurs), including any overt metastatic deposits, were dissected under a fluorescent stereomicroscope and the microRNA inserts amplified from the integrated retroviral genome by PCR.In an alternative approach, we have studied two populations of MDA-MB-231 human breast cancer cells that exhibit different behavior in vitro and in vivo to elucidate processes involved in cellular invasion and metastasis. A late-passage MDA-MB-231 (231variant) population formed invasive clusters when embedded in Matrigel, whereas early-passage (P3) MDA-MB-231 (231) cells did not. Moreover, 231v cells were more motile than 231 cells and showed enhanced tumorigenicity when grown as subcutaneous xenografts in nude mice, whereas proliferation and apoptotic response were indistinguishable between the two lines in vitro. Molecular characterization of the two cell populations cultured in 3D by array-based gene expression profiling revealed elevated expression of NF kappa B target genes in 231v cells (IL1A, IL1B, IL6, PTX3, NR4A2, HMOX1, FLIP). MicroRNA array analysis of 3D cultures also revealed marked induction (approximately 8-fold) of the related NF kappa B target microRNAs, mir-146a and mir-146b (Figure 1). Further evaluation of a panel of human breast cancer cell lines revealed that mir-146a and mir-146b were dramatically elevated in metastatic human breast cancer cell lines that display both epithelial-to-mesenchymal transition and high NF kappa B activity (MDA-MB-231, Hs578T, MDA-MB-436), compared to non-metastatic lines. Experiments to elucidate the function of mir-146a and mir-146b during breast cancer progression are underway. Figure 1: TaqMan qPCR analysis of mir-146a and mir-146b expression in 231 and 231v cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6142.

Phosphoglucose Isomerase/Autocrine Motility Factor Promotes Melanoma Cell Migration through ERK Activation Dependent on Autocrine Production of Interleukin-8

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Dynamic and Differential in Vivo Modifications of the Isoform HMGA1a and HMGA1b Chromatin Proteins

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.

A Potential Role for Interleukin-8 in the Metastatic Phenotype of Breast Carcinoma Cells

This study shows a strong correlation between the metastatic potentials of breast carcinoma cell lines and their ectopic expression of interleukin-8 (IL-8). Correlations exist for both constitutive and induced levels of IL-8 released. A correlation was also observed between cell morphology, metastatic potential, and IL-8 profile. Metastatic lines are fusiform in appearance, whereas, nonmetastatic lines are epithelioid. The metastatic potential of two breast carcinoma lines was examined using an orthotopic model of spontaneous metastasis. Metastatic cells formed rapidly growing, poorly differentiated primary tumors that metastasized. Nonmetastatic cells formed rapidly growing differentiated primary tumors that did not produce detectable metastases. Comparison of IL-8 expression by the parental cells and cell cultures developed from primary and metastatic tumors, demonstrates that IL-8 released by cultured cells from the primary tumor is higher than that of the parental cells, and IL-8 released by cultured cells derived from the metastatic lung tumors is greater than that released by cultured cells derived from the primary tumor. These data demonstrate a strong correlation between the metastatic phenotype of a cell and its IL-8 expression, suggesting a role for IL-8 in promoting the metastatic potential of breast tumor cells.

Metastatic sublines of an SV40 large T antigen immortalized human prostate epithelial cell line.

The available human prostate cancer cell lines that are metastatic in athymic nude mice all have complex, highly aneuploid karyotypes. Other prostatic cells immortalized by transforming genes of SV40 or HPV and converted to tumorigenicity by additional genetic manipulation are not reported to be metastatic. Tumorigenic sublines of human prostate epithelial cells previously immortalized by transfection with the SV40T antigen gene were obtained by sequential passage in male athymic nude mice. These sublines were evaluated histopathologically for tumorigenicity and metastasis in athymic nude mice after subcutaneous, intraperitoneal, and intraprostatic injection. Each subline was characterized by standard (GTG-banding) cytogenetic and FISH analysis, and RNase protection assays for androgen receptor expression. Two sublines produced metastases in lungs and the diaphragm of most mice after either intraprostatic or intraperitoneal injection. The M2205 subline formed large local tumors after intraprostatic injection. Cytogenetic aberrations present in the metastatic sublines, but not in the tumorigenic, nonmetastatic lines or the parental P69SV40T line, included dup(11)(q14q22), der(16) t (16;19) (q24;q13.1), which resulted in the loss of the short arm and proximal long arm of chromosome 19 (19q13.1-->19pter), and loss of the Y chromosome. None of the sublines expressed the androgen receptor. These cytogenetically defined, SV40T-immortalized human prostate epithelial cell lines, with distinct biological behaviors in vivo, provide additional tools for the genetic analysis of the emergence of metastatic capacity.

Analysis of the complete sequence of the novel metastasis-associated candidate gene, mtal, differentially expressed in mammary adenocarcinoma and breast cancer cell lines

To understand the genes and gene products involved in breast cancer invasion and metastasis, we previously isolated ten differentially expressed genes by differential cDNA library screening techniques, using the 13762NF rat mammary adenocarcinoma metastatic system. In this study, we further analysed a novel candidate metastasis-associated gene, mtal, previously designated clone 10.14. Northern blotting analyses showed that the steady-state mRNA expression level of mtal was fourfold higher in a highly metastatic line (MTLn3) than in a nonmetastatic line (MTC.4). The mtal gene was expressed at low levels in various normal rat organs, except testis, where it was expressed in high amounts. The mRNA expression levels of the human homologue of this gene were also examined in two human breast cancer metastatic systems; the ratios of mRNA were estimated to be MCF-7 (nonmetastatic):MCF7/LCC1 (invasive):MCF7/LCC2 (metastatic)= 1:2:4 and MDA-MB-468 (nonmetastatic):MDA231 (metastatic)= 1:4. Thus, the expression of this gene directly correlated with metastatic potential in two human systems, as well as in the rat metastatic system. Clone 10.14 was used to isolate a full-length cDNA clone for mtal, yielding the clone p10.14-C4.5, which was sequenced and analysed. Clone p10.14-C4.5 was 2756-bp long and contained a single open reading frame that could encode a protein of 703 amino acid (aa) residues. The aa sequence of mtal was found to be novel by database homology search and contained possible phosphorylation sites for tyrosine kinase, protein kinase C and casein kinase II. A Pro-rich stretch was found at the C-terminal end that completely matched the consensus sequence for the SH3-binding motif. Thus, the novel gene mtal may encode an important molecule that is functional in breast cancer invasion and metastasis, and could prove to be a useful probe to assess the malignancy of breast cancers.

Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines.

Thrombospondin (TSP) is a member of a family of extracellular matrix glycoproteins that may participate in multiple aspects of the metastatic cascade. We report an inverse correlation of steady-state Thbs-1 mRNA and protein expression with malignant progression among murine melanoma and human lung and breast carcinoma cell lines. Murine K-1735 melanoma cell lines of low metastatic potential, including K-1735 lines transfected with the murine nm23-1 cDNA, expressed higher TSP levels than related highly metastatic lines. In a model system of lung carcinoma malignant progression, immortalized human bronchial epithelial cells expressed higher TSP levels than v-Ki-ras, v-Ha-ras or n-ras transfectants, which in turn expressed higher TSP levels than tumor-derived, more aggressive variants. Among 3 unrelated breast carcinoma cell lines, Thbs-1 steady-state mRNA levels were greater in the 2 non-metastatic lines than the metastatic line. Our data show that malignant progression in some cell lines is associated with reduced TSP expression. The suppressive effects of nm23-1 transfection on metastatic potential are also associated with increased TSP expression; ras transfection, which results in increased tumorigenesis, is associated with decreased TSP expression.

Induction of fibroblast 92 kDa gelatinase/type IV collagenase expression by direct contact with metastatic tumor cells.

Previous studies have correlated release of the 92 kDa type IV collagenase/gelatinase by tumor cells in culture with metastatic potential. We have now demonstrated that the ability of tumor cells that do not express the 92 kDa gelatinase to induce release of this metalloproteinase from normal fibroblasts may also be associated with the metastatic phenotype. A transformed rat embryo cell line, 2.8, failed to release the 92 kDa gelatinase alone in culture, but gave rise to metastatic tumors whose explants contained the 92 kDa gelatinase. In contrast, a non-metastatic transformed cell line, RA3, did not express the 92 kDa gelatinase alone in culture or in tumor explants. To explore the mechanisms that might govern host-tumor cell interactions in this system, we have studied the effects of co-culture of these transformed cell lines with rat embryo fibroblasts (REF) in culture. 92 kDa gelatinase expression was induced by co-culture of 2.8 with REF, but co-culture of the non-metastatic line RA3 with REF did not result in induction of the 92 kDa gelatinase. The 92 kDa gelatinase in these co-cultures was released by the fibroblasts; methanol-fixed 2.8 cells induced 92 kDa gelatinase expression in REF, but fixed REF cells did not induce enzyme expression in 2.8 cells. This suggested that cell contact was required for induction, which was confirmed by showing that 92 kDa gelatinase induction in co-culture was abolished by separating REF from 2.8 by solute-permissive membranes. In addition, REF could not be stimulated to produce the 92 kDa gelatinase by 2.8-derived conditioned medium, by 2.8-derived extracellular matrix, or by isolated matrix components. These data indicate that metastatic tumor cells can induce 92 kDa gelatinase expression in fibroblasts through a mechanism dependent upon cell contact. In situ hybridization of nude mouse tumors derived from these transformed cell lines revealed 92 kDa gelatinase expression in the stroma of tumors from 2.8, but not in tumors from RA3. Therefore, the experiments based on in vitro co-culture of tumor cells and fibroblasts, together with the in situ localization of mRNA to host cells, suggest that host production of the 92 kDa gelatinase may occur in response to direct contact with metastatic tumor cells.