Expression In Metastatic Cells Research Articles

Immune surveillance of brain metastatic cancer cells is mediated by IFITM1.

Brain metastasis, most commonly originating from lung cancer, increases cancer morbidity and mortality. Although metastatic colonization is the rate-limiting and most complex step of the metastatic cascade, the underlying mechanisms are poorly understood. Here, in vivo genome-wide CRISPR-Cas9 screening revealed that loss of interferon-induced transmembrane protein 1 (IFITM1) promotes brain colonization of human lung cancer cells. Incipient brain metastatic cancer cells with high expression of IFITM1 secrete microglia-activating complement component 3 and enhance the cytolytic activity of CD8+ T cells by increasing the expression and membrane localization of major histocompatibility complex class I. After activation, microglia (of the innate immune system) and cytotoxic CD8+ T lymphocytes (of the adaptive immune system) were found to jointly eliminate cancer cells by releasing interferon-gamma and inducing phagocytosis and T-cell-mediated killing. In human cancer clinical trials, immune checkpoint blockade therapy response was significantly correlated with IFITM1 expression, and IFITM1 enhanced the brain metastasis suppression efficacy of PD-1 blockade in mice. Our results exemplify a novel mechanism through which metastatic cancer cells overcome the innate and adaptive immune responses to colonize the brain, and suggest that a combination therapy increasing IFITM1 expression in metastatic cells with PD-1 blockade may be a promising strategy to reduce metastasis.

Abstract PD9-07: Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer

Abstract Background: Luminal, i.e., estrogen receptor (ER)-positive breast cancer (BC) is a heterogeneous disease in terms of tumor progression, therapy response, and relapse. Additional biomarkers with a prognostic and predictive impact would facilitate advanced patient stratification and can reveal advanced therapeutic options for individual patients suffering from luminal BC subtype. First objective of this study was the hypothesis driven validation and identification of biomarkers associated with successful engraftment, augmented tumor growth, and enhanced metastasis upon xenotransplantation into HTM and non-humanized tumor mice. Second objective was the retrospective validation and correlation of aforementioned markers with clinical outcomes (disease free-survival [DFS] and overall survival [OS]) of luminal BC patients after neoadjuvant chemotherapy within the GeparTrio trial. Methods: Immunodeficient NSG mice with and without immunological humanization were transplanted with primary, ER-positive BC tissues and cells. Engraftment rates, tumor progression, and metastasis were monitored as a function of marker expression and genomic alterations, considered to be associated with tumor cell stemness and tumor initiating capacity. Functional assays were applied to demonstrate the impact of identified markers on tumor cell viability, growth and migration in-vitro. Dual color fluorescence-in-situ-hybridization to monitor mdm2 gene and the cen12 region was applied to 502 pretherapeutic ER-positive tissue specimens of BC patients treated with anthracycline/taxane based neoadjuvant chemotherapy within the GeparTrio trial. Mdm2 gene expression was analyzed in the entire luminal BC cohort as well as in luminal-A and luminal-B samples classified based on a Ki-67 cut-off of 20% (St. Gallen guideline). Associations with survival outcomes were studied by Cox regression models. Results: We observed an elevated CD44 and c-MET expression in metastatic cells compared to the primarily growing xenotransplantants. Moreover, we found mdm2 gene amplification was associated with tumor growth and pronounced metastatic potential in NSG mice. Functional assays unveiled a reduced viability, proliferation, and migration capacity of inherently mdm2 positive breast cancer cells upon mdm2 knock-down or anti-mdm2 targeting. Validation of mdm2 gain in luminal BC cohort within the GeparTrio trial revealed a significant association of mdm2 amplification with worse DFS (HR=1.80 [95%CI 1.16-2.79], log rank p=0.008) and OS (HR=1.75 [95%CI 1.00-3.05], log-rank p=0.047). This association was even stronger in luminal-A BC: DFS (HR=2.56 [95%CI 1.40-4.71], log rank p=0.002 and OS (HR=3.27 [95%CI 1.51-7.09], log-rank p=0.001). Conclusions: Mdm2 gene amplification facilitates ER-positive BC engraftment and progression in a preclinical in-vivo xenograft humanized NSG mouse model. Targeting mdm2 in-vitro reduced malignant cell propagation and growth. In addition, an increased mdm2 gene dose is strongly associated with an unfavorable outcome of luminal BC. Prospective studies are required to verify the suitability of mdm2 for advanced luminal BC stratification and therapeutic targeting. Citation Format: Anja Kathrin Wege, Valentina Vladimirova, Christine Solbach, Eva-Maria Rom-Jurek, Jens-Uwe Blohmer, Paul Jank, Bruno Sinn, Andreas Trumpp, Elisabetta Marangoni, Knut Engels, Wilko Weichert, Nicole Pfarr, Christoph Irlbeck, Bernhard Polzer, Olaf Ortmann, Marion van Mackelenbergh, Carsten Denkert, Sibylle Loibl, Gero Brockhoff. Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-07.

Abstract 2311: Identification of therapeutic targets in HNSCC

Abstract HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer, they can be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary HNSCC tumors and their respective metastatic sites/recurrences. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the data from the functional screens and to further characterize our cell line collection, we utilized several high through put approaches including mutational analysis, proteomics and gene expression profiling. Moreover, we performed chemical screens using libraries of over 4000 FDA approved drugs with the aim to combine our functional genomic data with the results from the drug screens to discover drug/gene “hit” combinations that would provide a basis for development of novel anti-cancer therapies.While HNSCC derived cell lines represent a valuable tool to study this disease, their biology might not always accurately represent the biology of the tumors they were derived from. Therefore, we looked at the expression of the components of several key pathways that we discovered using the in vitro characterization in a patient material from over HNSCC surgical samples constructed into a TMA. Five of the hits belonged to the Notch signalling pathway and 4 others came from the proteomic analysis as they showed higher levels of surface expression in metastatic cells and/or under hypoxic conditions. Interestingly, higher proportion of patients with late stages of HNSCC belonged to the high Jag2 and Hey1 populations as compared to patients with low stages of HNSCC that mostly expressed low or intermediate levels of these proteins. This data is in line with our observations that Jag2 and Hey1 expression is elevated in cells derived from metastatic sites and is low in cells derived from primary tumors. Proportion of cells with high CD66 and TRAIL proteins was also higher among the late stage tumors suggesting that these biomarkers are of interest for further investigation. We discovered several molecular pathways that are important in metastatic/hypoxic HNSCC. We are investigating our ability to target these pathways using FDA approved drugs in order to achieve durable cures. Citation Format: Maria Kondratyev, Aleksandra Pesic, Anna Dvorkin-Sheva, Troy Ketela, Natalie Stickle, Laurie Aiiles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Identification of therapeutic targets in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2311.

MiR-9 Does Not Regulate Lamin A Expression in Metastatic Cells from Lung Adenocarcinoma.

In lung adenocarcinoma, low lamin A expression in pleural metastatic cells has been proposed as a pejorative factor. miR-9 physiologically inhibits the expression of lamin A in neural cells and seems to be a central actor in the carcinogenesis and the metastatic process in lung cancer. Thus, it could be a good candidate to explain the reduction of lamin A expression in lung adenocarcinoma cells. miR-9 expression was analyzed in 16 pleural effusions containing metastatic cells from lung adenocarcinoma and was significantly reduced in patients from the ‘Low lamin A expression’ group compared to patients from the ‘High lamin A expression’ group. Then, carcinoma cells selection by fluorescence-activated cell sorting (FACS) was performed according to epithelial membrane antigen (EMA) expression, reflecting lamin A expression. miR-9 was underexpressed in lamin A− carcinoma cells compared to lamin A+ carcinoma cells in patients from the ‘Low lamin A expression’ group, whereas there was no difference of miR-9 expression between lamin A+ and lamin A− carcinoma cells in patients from the ‘High lamin A expression’ group. These results suggest that miR-9 does not regulate lamin A expression in metastatic cells from lung adenocarcinoma. On the contrary, miR-9 expression was shown to be reduced in lamin A-negative carcinoma cells.

Abstract B180: Effects of EMT process under MHC class I and TAP1 gene expression related to antigen presentation

Abstract Metastasis is a process that involves tumor cell migration from a primary tumor and its invasion to other tissues, which in turn are believed to be driven by a process called epithelial-mesenchymal transition (EMT). EMT process in cancer cells implies increase of malignance and metastatic potential by molecular and phenotypic modifications. MHC class I (MHC-I) is a protein complex used by cells for antigen presentation, specifically to T-cell CD8. It is believed that MHC-I gene expression may fluctuate along different cancer types, biologic processes and molecular modifications which cancer cells may suffer, and it could directly impact on system immunologic responses and T-cell-dependent immunotherapy treatment. This project aimed to evaluate MHC-I and TAP1 gene expression in nonmetastatic origin and metastatic origin cells under the effect of tumor growth factor beta (TGFb), a factor that induces EMT process. Three nonmetastatic cell lineages (A549, H1703, H23) and three metastatic cell lineages (H1792, H2023, H2030) of pulmonary carcinoma were cultured under controlled conditions in RPMI culture media supplemented with 10% of bovine fetal serum (BFS), 1% of antibiotics (penicillin and streptomycin), 2% of glutamine in incubator at 37oC and air atmosphere containing 5% of CO2. Cell cultures were supplemented or not with TGFb (4µg/mL) for 5 days to induce EMT process. After 5 days, cells were evaluated for acquiring mesenchymal cell morphology and MHC-I and TAP1 gene expression (relative quantification). Pool of cells was used to obtain RNA using RNAesy Extraction Kit (QIAGEN®) followed by RT-PCR reaction using High Capacity RNA-to-cDNA Kit (Applied Biosystems®) to obtain cDNA. Relative gene expression was analyzed using Real Time PCR using Fast SYBR™ Green Master Mix (Applied Biosystems®). Except by H23 cell, TGFb incubation showed to be effective and seems to induce EMT process within 5 days culture for both cell types. The cells acquired mesenchymal morphology characteristics such as elongation and increased size. It was also possible to identify a significant gene expression pattern for MHC-I and TAP1 gene expression between control and TGFb groups. Both MHC-I and TAP1 gene expression were shown to be upregulated in the majority of non-metastatic origin cells (A549, H1703, H23; p<0.05) and downregulated in metastatic origin cells (H1792, H2030, H2023; p<0.05). Indeed, nonmetastatic origin cells presented at least twice the expression of MHC-I compared to metastatic origin cells, and TAP1 presented nearly five times more expression in non-metastatic origin cells. Besides the cells apparently undergoing EMT process (morphologic aspects), and MHC-I and TAP1 gene expression differences between cell types, we did not observe such gene expression difference in both cell types when treated with TGFb. Only MHC-I gene expression showed to be affected by the TGFb and presented a slight decrease after 5 days incubation (p<0.05). However, such TGFb effect seems to affect only non-metastatic origin cells. Supposing metastatic origin cells, at some point, have suffered EMT process during metastasis process, it supports that downregulated MHC-I gene expression in metastasis cells could be due to the EMT process. Therefore, we conclude that TGFb was capable of inducing EMT process in both pulmonary carcinoma cell types and affects MHC-I and TAP1 genes expression in nonmetastatic origin cells. We also conclude that MHC-I and TAP1 gene expression are upregulated in nonmetastatic origin cells. Citation Format: Pedro Ratto Lisboa Pires, Pedro L.P. Xavier, Heidge Fukumasu. Effects of EMT process under MHC class I and TAP1 gene expression related to antigen presentation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B180.

Silencing of TGF-\u03b21 in tumor cells impacts MMP-9 in tumor microenvironment

Transforming growth factor (TGF)-β1 contributes to autocrine and paracrine functions in the tumor microenvironment (TME). The present study examined the effects of TGF-β1 crosstalk in TME and its role in mediating tumor formation and progression by targeted abrogation of TGF-β1 expression in metastatic cells in situ. Using species-specific primers, we found a significant increase in MMP-9 gene expression in the tumor-reactive stroma during late-stage metastasis in the lung. This effect was also confirmed in cancer-associated fibroblasts (CAFs) when co-cultured with the tumor cells. Knockdown of TGF-β1 expression in the tumor cells negatively affected matrix metalloproteinase (MMP)-9 gene expression. Fibroblasts, cultured in the presence of tumor cells with intact TGF-β1, showed a significant increase in proliferation rate, as well as expression of VEGF, bFGF, and SDF-1, which was not seen when TGF-β1 expression was abrogated in tumor cells. Absence of TGF-β1 in tumor cells also failed to result in myofibroblast differentiation. Co-implantation of CAFs and tumor cells with either intact TGF-β1 expression or devoid of TGF-β1 in vivo showed a significant increase in tumor growth kinetics in both cell types, suggesting a possible activation TGF-β receptor signaling in tumor cells in response to TGF-β from the TME.

P03.19 AQP4 in brain metastasis: its role and cross talk with the brain microenvironment.

AbstractAquaporins (AQPs) are a family of transmembrane channels, involved in transcellular and transepithelial fluids transport; AQP4 is enhanced in the brain, where is the main water channel, localized in the astrocytic endfoot processes. AQP4 is anchored to the blood-brain barrier (BBB) by the dystrophin-associated complex (DAPC) consisting of α-syntrophin and dystrophin. The AQP4 channel is co-expressed on the membrane with the inwardly rectifying K+ channel (Kir4.1) and the Na+/K+/Cl- cotransporter channel (NKCC1) to maintain extracellular homeostasis. In cancer AQPs promote cellular invasion and migration. Previously, in brain metastasis we observed AQP4 expression mostly in glial cells surrounding tumor, but also in metastatic cells (mainly on NSCLC). Therefore, in this study, our principal aim is to investigate the role of AQP4 pathway in tumor microenvironment, their cross talk with the extracellular matrix and its meaning in the metastatic process. So, we have performed a retrospective analysis about the expression of AQP4 and the molecules and channels associated to AQP4, on 87 brain metastases from different tumors: lung (n=36), breast (n=21), colon (n=7), kidney (n=11), melanoma (n=7), ovary (n=3), thyroid (n=1) and prostate (n=1). Dystrophin, α-syntrophin, Kir4.1 and NKCC1 were first evaluated by immunohistochemistry and then by molecular analysis. On 21 patients we analyzed both the primitive and metastatic tumor. We observed a great AQP4 expression at the metastasis-brain interface; high expression of α-syntrophin and NKCC1 was also observed, while Kir4.1 was lower. Western blot confirmed this expression of AQP4 both in tumor and in the surrounding tissue. qPCR showed a downregulation of AQP4 mRNA except for lung metastases. α-syntrophin showed a very high expression in metastatic cells compared to normal brain, but surprisingly mRNA levels were downregulated; dystrophin showed very low levels of expression both of protein and mRNA in tumor. NKCC1 and Kir4.1 channels were overexpressed compared to the brain. These results showed a deregulation of the whole pathway associated to AQP4 in the presence of the metastases. Besides, confocal images showed a polarization of AQP4 expression mostly in lung and breast metastases. The brain metastasis maintain the same profile of original tumor. Our preliminary studies in primitive neoplasms and their counterpart brain metastases suggest a cross talk from AQP4 pathway with adhesion molecules expression and extracellular matrix in response to the presence of tumor mass. In conclusion, our results confirmed a cross talk between AQP4 and the microenvironment during the metastatic process, having a role in to maintain homeostasis, trying to reduce the edema and to limit the spreading of tumor cells in the parenchyma. This study will provide an important step forward in the knowledge of microenvironment’s involvement in brain metastasis.

Stroma-induced Jagged1 expression drives PC3 prostate cancer cell migration; disparate effects of RIP-generated proteolytic fragments on cell behaviour and Notch signaling

The Notch ligand Jagged1 is subject to regulated intramembrane proteolysis (RIP) which yields a soluble ectodomain (sJag) and a soluble Jagged1 intracellular domain (JICD). The full-length Jagged1 protein enhances prostate cancer (PCa) cell proliferation and is highly expressed in metastatic cells. However, little is known regarding the mechanisms by which Jagged1 or its RIP-generated fragments might promote PCa bone metastasis. In the current study we show that bone marrow stroma (BMS) induces Jagged1 expression in bone metastatic prostate cancer PC3 cells and that this enhanced expression is mechanistically linked to the promotion of cell migration. We also show that RIP-generated Jagged1 fragments exert disparate effects on PC3 cell behaviour and Notch signaling. In conclusion, the expression of both the full-length ligand and its RIP-generated fragments must be considered in tandem when attempting to regulate Jagged1 as a possible PCa therapy.

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression.

The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined invitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of 'hot' spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer invitro and invivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

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.

Abstract 1145: Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor progression via Twist1 upregulation

Abstract p27 is a cell cycle regulator that acts largely to restrain normal cell growth. It is rarely mutated or deleted in human cancers, but is often degraded or mislocalized to the cytoplasm in aggressive tumors. Phosphorylation at T157 or T198 by different PI3K effector kinases leads to p27 cytoplasmic accumulation and enhanced cell motility and invasion in part via effects on the actin cytoskeleton, which are independent of its cell cycle role. However, the functional contributions of C-terminally phosphorylated, cytoplasmic p27 to cancer progression remain poorly understood. Targeted inhibition of PI3K/mTOR impaired tumor cell motility and metastasis via modulation of p27 in a bone metastatic model (Wander, S. et al. BCRT 2013). We observed that p27 knockdown in highly metastatic cancer lines with high levels of cytoplasmic p27pT157pT198 reverted EMT and impaired tumor cell invasion in vitro and metastasis in vivo. This led us to investigate whether PI3K-activated, phosphorylated p27 may function as a novel EMT regulator. A cell cycle defective (CK-) and double phosphomimetic p27 mutant (T157D/T198D or DD) was introduced into immortal human mammary epithelial cells with low PI3K activity. This revealed a novel, oncogenic function of p27 in regulating tumor progression, in that p27CK-DD induced epithelial-mesenchymal transition (EMT) and soft agar colony formation. p27CK-DD also increased motility, invasion and formation of metastasis in bladder and breast cancer models. A RT-PCR screen for potential p27-induced EMT mediators revealed a 20-fold increase in expression of the Twist1 transcription factor in p27CK-DD-transduced cells. Knockdown of Twist reversed the p27CK-DD-mediated increase in EMT marker expression. p27 knockdown rapidly attenuated Twist1-promoter activity and reduced Twist1 mRNA levels and also increased E-cadherin expression in metastatic cells, suggesting that Twist1 may play a critical role in p27CK-DD-induced EMT. These data extend our understanding of p27 function in human cancer and suggest that PI3K deregulated p27 may promote EMT and tumor metastasis through transcriptional activation of TWIST1. Citation Format: Dekuang Zhao, Alexandra H. Besser, Wen Zhou, Seth A. Wander, Jun Sun, Michael Durante, Feng Hong, Bin Wang, Tan Ince, Karoline Briegel, Joyce M. Slingerland. Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor progression via Twist1 upregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1145. doi:10.1158/1538-7445.AM2014-1145

Abstract B084: EMT-inducing transcription factors Twist1 and Snail1 non-cell autonomously increase metastatic properties of non-Twist1 or Snail1 expressing cells via induction of Six1

Abstract Breast cancer is the second leading cause of cancer related deaths in women and more than 90% of these deaths result from metastatic disease rather than from the primary tumor burden. The primary tumor itself is a heterogeneous mass of cells. Thus a better understanding of metastasis and cancer cell dynamics within the primary tumor will aid in developing efficient strategies to increase patient survival. Twist1 and Snail1 are two epithelial to mesenchymal transition (EMT)-inducing transcription factors (TF) that are known to increase metastasis through mechanisms that are cell autonomous. Our data demonstrate that these TFs can also non-cell autonomously increase in-vitro invasion and migration, as well as alter the expression of numerous genes and proteins associated with EMT, when conditioned medium (CM) taken from cells expressing these TFs is placed on cells not expressing the TFs. We show that the non-cell autonomous effects of Twist1 and Snail1 are in part induced via the ability of these TFs to up-regulate another known EMT related transcription factor, Six1. Indeed, we show that loss of Six1 downstream of these TFs mitigates their ability to non-cell autonomously influence EMT related characteristics including invasion and migration of non-metastatic non-TF expressing cells in-vitro, indicating that Six1 is a key player downstream of these TFs. Overexpression and knockdown of Six1 itself, in multiple mouse and human breast carcinoma cell lines non-cell autonomously influences invasion, migration, anoikis resistance, and alters the expression of EMT-related genes in the non-metastatic non-Six1 expressing cells in-vitro. We also show that Six1 expression in metastatic cells is sufficient to non-cell autonomously increase the metastatic potential of weakly metastatic, non Six1-expressing cells, when these cells are fluorescently tagged and co-injected into mice. We have identified several pro-metastatic cytokines, including IL-6 and Osteopontin, that are not only increased in CM from cells expressing Six1, but also have increased expression in cells expressing Twist1 and Snail1, in a manner that is dependent on Six1. We are currently assessing whether these cytokines play a role in the ability of Twist1, Snail1 and Six1 to mediate invasion, migration as well as metastasis of neighboring cells that do not express these EMT-inducing TFs. Importantly, EMT related transcription factors such as Six1, Twist1 and Snail1 are associated with poor prognosis and increased metastasis in breast cancer as well as in other cancers. Therefore, it is critical that we understand not only how these TFs mediate aggressive phenotypes in cells expressing these factors, but also how they mediate non-cell autonomous effects that contribute to increased metastasis of the neighboring non-TF expressing cells within the heterogeneous tumor. Citation Format: Deepika Neelakantan, Ritsuko Iwanaga, David J. Drasin, Heide L. Ford. EMT-inducing transcription factors Twist1 and Snail1 non-cell autonomously increase metastatic properties of non-Twist1 or Snail1 expressing cells via induction of Six1. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B084.

Metastasis suppression by BRMS1 associated with SIN3 chromatin remodeling complexes

Epigenetic regulation of gene transcription by histone modification and chromatin remodeling has been linked to many biological and pathological events including cancer metastasis. Breast cancer metastasis suppressor 1 (BRMS1) interacts with SIN3 chromatin remodeling complexes, and, upon forced expression in metastatic cells, a nearly complete suppression of metastasis is noted without preventing primary tumor growth. The data for BRMS1-mediated metastasis suppression and SIN3 interaction are clear; however, connecting the inhibition directly to the association of BRMS1 with SIN3 complexes is currently not well defined. Considering the recent advancements in developing epigenetic drugs for cancer therapy, an improved understanding of how the interactions between BRMS1 and SIN3 regulate the process of metastasis should lead to novel therapies specifically targeting the most deadly aspect of tumor progression. In this article, the data for BRMS1-mediated metastasis suppression are reviewed with a focus on how the SIN3 chromatin remodeling complexes may be functionally involved.

Ezrin Expression as a Prognostic Marker in Colorectal Adenocarcinoma

Ezrin protein acts in the regulation of cytoskeletal and directly influences survival and tumor progression; there is an increase in its expression in metastatic cells and tissues in several types of cancer including colorectal cancer. 250 Patients with colorectal cancer submitted to surgery from 1995 to 2002. Protein expression was carried through by Tissue Micro Array immunohistochemical tests of paraffined neoplasic tissues and associated with clinical variables. Differentiation degree, lymph node invasion, metastasis at diagnosis, and palliative surgery were associated to a higher expression of the protein and survival. Higher expression of the Ezrin correlates with tumor aggressiveness and worse prognosis for colorectal cancer.

T‐lymphocyte maturation‐associated protein gene as a candidate metastasis suppressor for head and neck squamous cell carcinomas

Previous gene expression profiles revealed the T-lymphocyte maturation-associated protein (MAL) gene as being frequently downregulated in head and neck cancer. To define the relationship between the MAL gene and the metastatic process, we evaluated the expression status of the gene in matched primary and metastatic tumors of head and neck cancer by semiquantitative reverse transcription-polymerase chain reaction. Furthermore, we aimed to identify potential genetic and epigenetic mechanisms associated with downregulation of MAL, including loss of heterozygosity (LOH), mutation, and hypermethylation. Thirty-five cell lines of University of Turko squamous cell carcinoma (UT-SCC) series derived from head and neck cancer, including nine pairs from matched primary and metastatic tumors, and 30 pairs of matched primary and metastatic tumor samples were analyzed. Twenty out of 35 (57%) cell lines showed downregulation of MAL expression, whereas no expression was found in 10 cell lines (29%). Considering matched primary and metastatic tumor-derived cell-line pairs, four pairs showed decreased expression only in metastasis-derived cells compared with their primary counterparts. Expression analysis of 21 tissue samples demonstrated decreased or no expression of MAL mRNA in 43% of metastatic tumors compared with matched primary tumors. Relating to mechanisms of downregulation, LOH was observed in 30% of primary tumors and 38% of their metastatic counterparts by a MAL-specific microsatellite marker. Furthermore, we found restoration of MAL mRNA after treatment with demethylating agent (5-aza-2'-deoxycytidine) in 9 (45%) out of 20 cell lines. No mutation was found in UT-SCC cell lines. In conclusion, our findings indicate selective downregulation of MAL expression in metastatic cells, suggesting the MAL gene as a new metastasis-suppressor candidate for head and neck cancer. LOH and hypermethylation appeared to be important mechanisms for inactivation of MAL function.

Nitric Oxide Mediates Natural Polyphenol-induced Bcl-2 Down-regulation and Activation of Cell Death in Metastatic B16 Melanoma

Intravenous administration to mice of trans-pterostilbene (t-PTER; 3,5-dimethoxy-4'-hydroxystilbene) and quercetin (QUER; 3,3',4',5,6-pentahydroxyflavone), two structurally related and naturally occurring small polyphenols, inhibits metastatic growth of highly malignant B16 melanoma F10 (B16M-F10) cells. t-PTER and QUER inhibit bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. However, the molecular mechanism(s) linking polyphenol signaling and bcl-2 expression are unknown. NO is a potential bioregulator of apoptosis with controversial effects on Bcl-2 regulation. Polyphenols may affect NO generation. Short-term exposure (60 min/day) to t-PTER (40 microM) and QUER (20 microM) (approximate mean values of the plasma concentrations measured within the first hour after intravenous administration of 20 mg of each polyphenol/kg) down-regulated inducible NO synthetase in B16M-F10 cells and up-regulated endothelial NO synthetase in the vascular endothelium and thereby facilitated endothelium-induced tumor cytotoxicity. Very low and high NO levels down-regulated bcl-2 expression in B16M-F10 cells. t-PTER and QUER induced a NO shortage-dependent decrease in cAMP-response element-binding protein phosphorylation, a positive regulator of bcl-2 expression, in B16M-F10 cells. On the other hand, during cancer and endothelial cell interaction, t-PTER- and QUER-induced NO release from the vascular endothelium up-regulated neutral sphingomyelinase activity and ceramide generation in B16M-F10 cells. Direct NO-induced cytotoxicity and ceramide-induced mitochondrial permeability transition and apoptosis activation can explain the increased endothelium-induced death of Bcl-2-depleted B16M-F10 cells.

Association between Pterostilbene and Quercetin Inhibits Metastatic Activity of B16 Melanoma

Inhibition of cancer growth by resveratrol (trans-3,5,4'trihydroxystilbene; RESV), a phytoalexin present in many plant species, is limited by its low bioavailability. Pterostilbene (3,5-dimethoxy-4'-hydroxystilbene; PTER) and quercetin (3,3',4',5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols, show longer half-life in vivo. In vitro growth of highly malignant B16 melanoma F10 cells (B16M-F10) is inhibited (56%) by short-time exposure (60 min/day) to PTER (40 μM) and QUER (20 μM) (approximate mean values of plasma concentrations measured within the first hour after intravenous administration of 20 mg/kg of each polyphenol). Intravenous administration of PTER and QUER (20 mg/kg per day) to mice inhibits (73%) metastatic growth of B16M-F10 cells in the liver, a common site for metastasis development. The antimetastatic mechanism involves: 1) a PTER-induced inhibition of vascular adhesion molecule 1 expression in the hepatic sinusoidal endothelium, which consequently decreases B16M-F10 cell adhesion to the endothelium through very late activation antigen 4; and 2) a QUER- and PTER-induced inhibition of Bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. Our findings demonstrate that the association of PTER and QUER inhibits metastatic melanoma growth and extends host survival.

Differential expression of neutral endopeptidase-24.11 (neprilysin) and endothelin-converting enzyme in human prostate cancer cell lines.

Neutral endopeptidase-24.11 (neprilysin; NEP/CD10) is a cell surface metallopeptidase expressed by prostatic epithelial cells that degrades various bioactive peptides including endothelin. Endothelin-converting enzyme (ECE), the key enzyme of endothelin biosynthesis, catalyses the final processing step in the pathway. Neuropeptide substrates of NEP, including endothelin, have been implicated in the growth of androgen-independent prostate cancer. We have surveyed the expression of NEP and ECE in a range of prostate cancer cell lines. Western analysis reveals that ECE-1 is expressed abundantly in all the malignant cell lines tested, except for LNCaP. In contrast, LNCaP cells express high levels of NEP, while NEP was not detected in PC-3, DU145 and other metastatic cell lines that were tested. Of the normal immortalized prostate epithelial cell lines, PNT1a shows equivalent amounts of NEP and ECE. PNT2-C2 shows poor NEP expression but an abundance of ECE. P4E6, by comparison, has low levels of both ECE and NEP. These differences in expression may render these cell lines useful in experimental models for future study. Benign prostatic hyperplasia primary epithelial cells express much higher levels of NEP than malignant primary epithelial cells, but neither show ECE expression. On the other hand, surrounding stromal cell populations have detectable ECE levels. An absence of ECE in malignant and benign prostatic hyperplasia cells of primary epithelial origin suggests an important role for stromal interaction and paracrine production of ECE within the host. The upregulation of ECE expression in metastatic cells in culture may be indicative of its role in metastatic progression. A differential profile of ECE and NEP could contribute to an abundance of mitogenic peptides aiding the progression of androgen-independent prostate cancer.

Staging of head and neck squamous cell carcinoma using theMET oncogene product as marker of tumor cells in lymph node metastases

In head and neck squamous cell carcinomas (HNSCC), metastasis to cervical lymph nodes is a major determinant of patient outcome. To detect metastases, we used the MET oncogene as marker, which encodes the receptor for hepatocyte growth factor/scatter factor, mediating epithelial cell motility and invasiveness. The MET gene is expressed in epithelia and over-expressed in carcinomas of specific histotypes, but not in lymphatic tissue. A total of 151 lymph nodes from 20 squamous cell carcinomas were studied with both in-depth histology and end-point and real-time quantitative RT-PCR. MET-encoded sequences were found in 61 of 151 nodes (40%), of which 24 (16%) were found metastatic by in-depth histopathology. Parallel routine histopathologic analysis of 654 lymph nodes from the same cases identified 36 metastases (5%). Real-time quantitative RT-PCR was used to measure MET gene-specific mRNA in normal tissues, primary tumors and lymphatic metastases and showed a 2-8-fold increased expression in tumor cells which metastasize. RT-PCR for 3 cytokeratins expressed in HNSCC (K4, K10 and K13) proved to be less sensitive in detecting occult lymphatic metastases. Western blot analysis demonstrated the presence of the full-size MET receptor in primary tumors and lymph node metastases; immunohistochemistry showed receptor localization in tumor cells. Altogether, these data demonstrate that the MET gene product is a valuable marker with which to detect occult tumor cells in lymph nodes, thanks to its high expression in metastatic cells. After RT-PCR analysis we were able to attribute a more advanced stage to 10 out of 20 HNSCC cases, including 5 cases classified as tumor-free after routine histopathology.

Molecular changes in human melanoma metastasis.

The molecular changes associated with the transition of melanoma cells from radial growth phase to vertical growth phase (metastatic phenotype) are not well defined. Our recent studies have demonstrated that the two tumor suppressor genes, p53 and p16/CDKN2, do not play a major role in the acquisition of the metastatic phenotype in human melanoma. Mutations in p53 are infrequent and do not correlate with the metastatic potential of human melanoma cells while p16/CDKN2 abnormalities are frequent, but are not pre-requisite for the acquisition of the metastatic phenotype. On the other hand, the tyrosine-kinase receptor c-KIT and the cell adhesion molecule MCAM/MUC-18 play active roles in the progression of human melanoma. Metastatic melanoma cells overexpress MCAM and do not express the c-KIT receptor. Enforced c-KIT expression in metastatic cells significantly inhibited their growth and metastatic potential in nude mice. Furthermore, exposure of c-KIT-positive melanoma cells in vitro and in vivo to stem cell factor (SCF), the ligand for c-KIT, triggered apoptosis of these cells but not of normal melanocytes. Ectopic expression of MCAM into primary cutaneous melanoma cells enhanced their tumorigenicity and metastatic ability in vivo. We found that both genes, c-KIT and MCAM, are regulated by the transcription factor AP-2 and that metastatic melanoma cells do not express AP-2. We therefore propose that loss of AP-2 might be a crucial event in the progression of human melanoma.