Cancer Metastatic Potential Research Articles

Abstract 1476: Cell-free miR-141 as a molecular marker for prostate cancer metastasis

Abstract Prostate cancer is the second most commonly diagnosed neoplasia in men. Currently, there is no available non-invasive tool to assist in the identification of the aggressive form of this disease. Cell-free nucleic acid has been tested as a potential new molecular tool that can be useful in the diagnosis and prognosis of prostate cancer. MicroRNAs are a class of non-coding RNAs, that are shown to impact the development and progression of prostate cancer and their detection in plasma have been indicated as a promising non-invasive tool for disease screening and prognosis assessment. Among the several deregulated miRNAs that are observed in prostate cancer, the miRNA-141-3p was observed with upregulated expression levels in prostate cancer, particularly in the metastatic lesions. Therefore, the main aim of this study was to verify the diagnostic potential of miR-141 as a circulating tumor marker for prostate cancer and determine its functional role in modulating tumorigenesis in prostate cancer metastatic cell lines. MiR-141 expression analysis was performed in plasma samples of 102 prostate cancer patients without treatment and of 50 health controls by RT-qPCR. The experimental assays were performed by transfecting the PC3 cell line using miRNA-141 mimic/ inhibitor systems and directly accessing their effect in classical tumor phenotypes. Expression of the miR-141 were found significantly upregulated in the metastatic patients’ plasma specimens (FC=9.11, p=0.04; AUC=0.66) when compared to the patients without metastasis, showing its screening potential for prostate cancer metastasis. The in vitro ectopic expression of miR-141 in PC3 cell line showed a significant increased in cell proliferation, associated with changes in cell cycle (decreased number of cells in the G1 phase). Conversely, the inhibition of miR-141 showed an increase in the migratory ability, cell adhesion and in the Docetaxel cytotoxicity of the PC3 cells. Western-blot analysis showed that the inhibition of the miR-141 levels reduced the expression of the EMT inducers markers E-CADHERIN and CLAUDIN, and up-regulated the levels of ZEB-1, pAKT and VIMENTIN, consistent with their repression roles in the EMT process. These findings showed that miR-141 presents a direct function in controlling some of the in vitro metastatic phenotypes in prostate cancer, through the regulation of the epithelial-mesenchymal transition expression proteins. In conclusion, miR-141 presents a potential use as a minimally invasive molecular marker for prostate cancer diagnosis and can be used as a druggable target for therapy, particularly in the metastatic clinical setting. Citation Format: Marilesia Ferreira de Souza, Ilce Mara de Syllos Cólus, Aline Simoneti Fonseca, Hellen Kuasne, Paulo Emilio Fuganti, Deepak Kumar, Silvia Regina Rogatto, Luciane Regina Cavalli. Cell-free miR-141 as a molecular marker for prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1476. doi:10.1158/1538-7445.AM2017-1476

Abstract 467: Analysis of human hypoxia related miRNAs in MDM2 transfected prostate cancer cells

Abstract Recent studies have shown the crucial role of miRNAs in regulating cancer initiation, progression and metastatic process via influencing gene expression patterns. However, the distinct mechanism by which miRNA regulates tumorigenesis remains as an area requiring detailed research investigations. In our earlier studies, we have demonstrated a positive correlation between MDM2 expression and hypoxia inducible factor -1alpha (HIF-1α) levels in both normoxic and hypoxic conditions using prostate cancer cell lines. Subsequently, we postulated that the upregulation of HIF-1α is most likely regulated by MDM2 in cancers cells that are positive for this gene expression. Hence, to confirm our hypothesis, we utilized miScript miRNA PCR Array analysis (Qiagen, USA) and compared the gene expression profile of the hypoxia signaling pathway in LNCaP (prostate cancer cells) and LNCaP-MST (MDM2 transfected prostate cancer cells) under normoxic conditions. This study was expected to outline how MDM2 impacts the differential expression of miRNAs and leading to enhancement of tumor angiogenesis via increase in HIF-1α or other genes of the hypoxia signaling pathway. Our preliminary data indicate that hsamiR99a5p, hsamiR125b5p, hsamiR1385p, hsamiR199b5p and hsalet7c5p are significantly upregulated in LNCaP-MST cells compared to LNCaP. In addition, some of the miRNAs such as hsalet7d5p, hsalet7e5p, hsalet7f5p, hsalet7g5p and hsalet7i5p are significantly downregulated in the same cells. Among the upregulated miRNAs, hsamiR125b5p emerged as an important gene that is known to downregulate the protein levels of hypoxia inducible factor 1alpha inhibitor (HIF1AN or FIH), which can normally inhibit the transcriptional activity of HIF-1α in both normoxic and hypoxic conditions. Therefore, overexpression of hsamiR125b5p seems to be associated with the increase in HIF-1α level. From the aforementioned findings, it is confirmed that the MDM2 overexpression is impacting miRNA levels such as hsamiR125b5p that are involved in hypoxia related signaling pathway. Thus, our results offer a convincing evidence that MDM2 can promote angiogenesis and increase metastatic potential of cancers through altering the miRNA expression levels. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Saad Ebrahim Alobid, Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Appu Rathinavelu. Analysis of human hypoxia related miRNAs in MDM2 transfected prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 467. doi:10.1158/1538-7445.AM2017-467

Cancer stemness and metastatic potential of the novel tumor cell line K3: an inner mutated cell of bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) transplantation has been used for therapeutic applications in various diseases. Here we report MSCs can malignantly transform in vivo. The novel neoplasm was found on the tail of female rat after injection with male rat bone marrow-derived MSCs (rBM-MSCs) and the new tumor cell line, K3, was isolated from the neoplasm. The K3 cells expressed surface antigens and pluripotent genes similar to those of rBM-MSCs and presented tumor cell features. Moreover, the K3 cells contained side population cells (SP) like cancer stem cells (CSCs), which might contribute to K3 heterogeneity and tumorigenic capacity. To investigate the metastatic potential of K3 cells, we established the nude mouse models of liver and lung metastases and isolated the corresponding metastatic cell lines K3-F4 and K3-B6. Both K3-F4 and K3-B6 cell lines with higher metastatic potential acquired more mesenchymal and stemness-related features. Epithelial-mesenchymal transition is a potential mechanism of K3-F4 and K3-B6 formation.

Preclinical study of c-Met inhibitor as an adjuvant treatment for hepatocellular carcinoma with macroscopic portal vein invasion.

301 Background: Patients with advanced hepatocellular carcinoma (HCC) demonstrating a macroscopic portal vein tumor thrombus (PVTT) have been reported to have an extremely poor prognosis. Palliative sorafenib is the only recommended treatment option. Methods: We statistically compared the patient characteristics and surgical outcomes in HCC patients with PVTT. Among 1,611 hepatic resections, 105 cases of PVTT were identified. Microarray analysis was performed in three patients to identify gene expression changes in PVTT compared with those in the principal tumor, and the changes were validated in 20 human HCC tissues with PVTT. The human HCC cell lines HuH-7 and SKHep-1 were used for this experimental study. A subcutaneously transplanted xenograft model was employed for the in vivo study. The c-Met inhibitor SU11274 was used in both in vitro and in vivoanalyses. Results: The median survival time in patients with PVTT was 2.01 years, while that in patients without PVTT was 6.43 years; the median time to recurrence was 0.31 and 1.61 years, respectively. Microarray analysis revealed 36 genes related to PVTT. Immunohistochemistry analysis revealed that compared with the principal tumor, E-cadherin (a key regulator of cancer metastatic potential) significantly decreased in PVTT in all 20 patients. The c-Met inhibitor elevated the E-cadherin expression level in HCC cells both in vitro and in vivo. This inhibitor induced sheet formation and attenuated the migration of HCC cells. Conclusions: Although liver resection provides acceptable overall survival for patients with PVTT, the recurrence rate remains high. The c-Met inhibitor exhibits an anti-metastatic effect in vitro and in vivo and may be useful as an adjuvant treatment for PVTT.

PSMA-Targeted Theranostic Nanocarrier for Prostate Cancer.

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin β1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin β1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size.

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Dietary Flavonoids Luteolin and Quercetin Suppressed Cancer Stem Cell Properties and Metastatic Potential of Isolated Prostate Cancer Cells.

A highly invasive Du145-III subline was isolated by three successive passages of the parental Du145 prostate tumor cell line (Du145-P) through a Boyden chamber with matrigel-coated membrane support. Du145-III cells showed great invasion potential based on their increased ability to spread/migrate and enhanced expression/secretion of the matrix metalloproteinase 9 (MMP9). Du145-III cells exerted vasculogenic mimicry (VM) properties, reminiscent of endothelial cell characteristics and expressed elevated levels of cancer stem cell (CSC) markers, including Nanog, Sox2, CD44 and ABCG2 and ability to self-renew. Of prominence, MMP9 was required for the induction of VM and for increased stemness in Du145-III cells. Using Du145-III as a model, the effects of dietary flavonoids, luteolin and quercetin, were evaluated on stemness and invasion capacity of Du145-III cells in relation to JNK signaling pathway activation. These flavonoids depressed the malignancy of highly invasive Du145-III cells, VM, anchorage-independent spheroid formation and expression of certain CSC markers. Since luteolin and quercetin were able to target CSC cells and prevent cancer cell invasiveness, may serve as potential anti-angiogenesis and anti-metastasis agents.

Kinesin-5 Contributes to Spindle-length Scaling in the Evolution of Cancer toward Metastasis.

During natural evolution, the spindles often scale with cell sizes to orchestrate accurate chromosome segregation. Whether in cancer evolution, when the constraints on genome integrity are relaxed, cancer cells may evolve the spindle to confer other advantages has not been investigated. Using invasion as a selective pressure in vitro, we found that a highly metastatic cancer clone displays a lengthened metaphase spindle, with faster spindle elongation that correlates with transiently elevated speed of cell migration. We found that kinesin-5 is upregulated in this malignant clone, and weak inhibition of kinesin-5 activity could revert the spindle to a smaller aspect ratio, decrease the speed of spindle pole separation, and suppress post-mitotic cell migration. A correlation was found between high aspect ratio and strong metastatic potential in cancers that evolved and were selected in vivo, implicating that the spindle aspect ratio could serve as a promising cellular biomarker for metastatic cancer clones.

PlexinD1 Is a Novel Transcriptional Target and Effector of Notch Signaling in Cancer Cells

The secreted semaphorin Sema3E controls cell migration and invasiveness in cancer cells. Sema3E-receptor, PlexinD1, is frequently upregulated in melanoma, breast, colon, ovarian and prostate cancers; however, the mechanisms underlying PlexinD1 upregulation and the downstream events elicited in tumor cells are still unclear. Here we show that the canonical RBPjk-dependent Notch signaling cascade controls PlexinD1 expression in primary endothelial and cancer cells. Transcriptional activation was studied by quantitative PCR and promoter activity reporter assays. We found that Notch ligands and constitutively activated intracellular forms of Notch receptors upregulated PlexinD1 expression; conversely RNAi-based knock-down, or pharmacological inhibition of Notch signaling by gamma-secretase inhibitors, downregulated PlexinD1 levels. Notably, both Notch1 and Notch3 expression positively correlates with PlexinD1 levels in prostate cancer, as well as in other tumor types. In prostate cancer cells, Sema3E-PlexinD1 axis was previously reported to regulate migration; however, implicated mechanisms were not elucidated. Here we show that in these cells PlexinD1 activity induces the expression of the transcription factor Slug, downregulates E-cadherin levels and enhances cell migration. Moreover, our mechanistic data identify PlexinD1 as a pivotal mediator of this signaling axis downstream of Notch in prostate cancer cells. In fact, on one hand, PlexinD1 is required to mediate cell migration and E-cadherin regulation elicited by Notch. On the other hand, PlexinD1 upregulation is sufficient to induce prostate cancer cell migration and metastatic potential in mice, leading to functional rescue in the absence of Notch. In sum, our work identifies PlexinD1 as a novel transcriptional target induced by Notch signaling, and reveals its role promoting prostate cancer cell migration and downregulating E-cadherin levels in Slug-dependent manner. Collectively, these findings suggest that Notch-PlexinD1 signaling axis may be targeted to impair prostate cancer cell invasiveness and metastasis.

The truncated somatostatin receptor sst5TMD4 stimulates the angiogenic process and is associated to lymphatic metastasis and disease-free survival in breast cancer patients.

The truncated somatostatin receptor sst5TMD4 is associated with poor prognosis in breast cancer and increases breast cancer cell malignancy. Here, we examined the cellular/molecular mechanisms underlying this association, aiming to identify new molecular tools to improve diagnosis, prognosis or therapy. A gene expression array comparing sst5TMD4 stably-transfected MCF-7 cells and their controls (empty-plasmid) revealed the existence of profound alterations in the expression of genes involved in key tumoral processes, such as cell survival or angiogenesis. Moreover, sst5TMD4-overexpressing MCF-7 and MDA-MB-231 cells demonstrated increased expression/production of pro-angiogenic factors and enhanced capacity to form mammospheres. Consistently, sst5TMD4-expressing MCF-7 cells induced xenografted tumors with higher VEGF levels and elevated number of blood vessels. Importantly, sst5TMD4 was expressed in a subset of breast cancers, where it correlated with angiogenic markers, lymphatic metastasis, and reduced disease-free survival. These results, coupled to our previous data, support a relevant role of sst5TMD4 in the angiogenic process and reinforce the role of sst5TMD4 in breast cancer malignancy and metastatic potential, supporting its possible utility to develop new molecular biomarkers and drug therapies for these tumors.

Clinical Significance of Histone Demethylase NO66 in Invasive Colorectal Cancer.

Targeting epigenetic regulators is a promising therapeutic strategy against cancer. However, because of the broad spectrum of targets, selective inhibition of cancer-associated genes remains a major challenge. To address this issue, we focused on the oncogene-regulated histone demethylase, nucleolar protein 66 (NO66 [C14orf169/MAPJD]), which is known to work coordinately with the well-characterized oncogene, c-MYC. To investigate expression patterns and clinical significance of NO66 in colorectal cancer (CRC), we performed immunohistochemical staining in 114 CRC cases. We performed functional analysis of NO66 to evaluate its contribution to proliferation and migration ability in CRC cells in vitro. NO66 was selectively expressed in CRC tissues. Furthermore, high expression levels of NO66 were associated with cancer metastatic potential, including lymphatic duct invasion (p=0.047), venous invasion (p=0.033), and lymph node metastasis (p=0.015). Multivariate analysis indicated that NO66 was an independent prognostic factor for overall survival. In vitro assays revealed that NO66 expression is closely associated with malignant potential, including proliferation, migration and anti-apoptotic activity. NO66 is an independent prognostic factor in CRC. The cancer-selective expression patterns and its involvement in metastatic phenotypes suggest that NO66 is not only a crucial biomarker but is also a promising therapeutic target in CRC.

Abstract 1858: Analyzing the impact of MDM2 overexpression on cell cycle pathway-related genes using polymerase chain reaction array

Abstract The human homologue of the mouse double minute 2 (MDM2) oncogene has been well characterized as a negative regulator of the tumor suppressor p53. It has been established that p53 regulates genes that are involved in cell cycle arrest, apoptosis, DNA repair and senescence. MDM2 interacts primarily with p53 in an autoregulatory negative feedback loop and targets it for ubiquitylation and proteasomal degradation there by attenuating p53 cell cycle arrest and apoptosis functions. Studies have shown that MDM2 also exerts its effects via p53 independent pathways. These p53-independent functions of MDM2 may have a role in cancer etiology and progression, yet the exact function and significance of these interactions are not fully understood. MDM2 is overexpressed in more than 40 different types of malignancies, including wide varieties of carcinomas and sarcomas, which is believed to be associated with worse clinical prognosis and increased likelihood of distant metastases. Hence, MDM2 is proving to be a key player in human cancer and thus an important target for therapeutic intervention. In this study, we aimed to investigate the effects of MDM2 overexpression on cell cycle pathways by using LNCaP prostate cancer cells and MDM2 transfected LNCaP-MST. The LNCaP-MST cells expressed at least 10-folds higher levels of MDM2 mRNA due to transfection. We used the Human Cell Cycle PCR array to compare the gene expression profiles of LNCaP and LNCaP-MST cells. Our experimental results clearly indicated that MDM2 modulates the expression of cell cycle related genes. The following genes were found to be significantly down-regulated in LNCaP-MST cells compared to LNCaP: CCND2, GADD45A, RB1, RBBP8, CDKN1A and TP53. Genes such as BIRC5, CDK1 were found to be significantly up-regulated. Heat map and scatter plot analysis further confirmed the alterations in the expression levels of these genes following the MDM2 overexpression. In addition, cluster analysis further denoted that the gene expression patterns positively correlated with cell proliferation and tumor growth. Our experimental results offer conclusive evidence that MDM2 can impact the cancer growth and metastatic potential by altering the gene expressions in the cell cycle pathway. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged). Citation Format: Priya Dondapati, Thiagarajan Venkatesan, Appu Rathinavelu. Analyzing the impact of MDM2 overexpression on cell cycle pathway-related genes using polymerase chain reaction array. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1858.

Abstract 2493: Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target in breast cancer

Abstract Introduction: Breast cancer is a heterogeneous disease, and is comprised of a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we targeted DCLK1 as a novel therapeutic strategy in breast cancer cells to reduce cancer cell self-renewal, tumorigenesis and metastasis. Methods: Anti-DCLK1-siRNA was transfected into breast cancer cell lines (MCF7 [luminal] and MDAMB231 [basal-triple negative breast cancer cell line]). For analysis of circulating tumor cells, MDAMB231/MCF7 cells were stably transfected with RFP (red fluorescence protein). Tumor cell colony formation, mammosphere formation (self-renewal), cell migration and invasion were assessed to evaluate the effect of DCLK1 knockdown and or DCLK1 overexpression on breast cancer cells self-renewal and metastatic potential. Xenograft model was used to identify the role of DCLK1 on breast cancer cell growth and circulating tumor cells in vivo. The data were generated utilizing experimental protocols for clonogenic culture, circulating tumor cells, immunohistochemistry, RT-qPCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal breast tissue. Silencing DCLK1 via RNA interference decreased the colony formation (∼70%), and self-renewal ability (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to less (40-80%) breast cancer cells migration and invasion. Furthermore, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were markedly reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in xenograft tumors resulted in reduced tumor growth (∼81%) and CTCs (∼95%) in vivo. However, overexpression of DCLK1 in the non-metastatic MCF7 breast cancer cell line increased its in vitro migration and invasion potency and pluripotency along with in vivo tumor formation and progression. Conclusions: DCLK1 expression is associated with breast cancer cell self-renewal, metastasis and tumorigenesis. Targeting DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis and tumor growth. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Dongfeng Qu, William Berry, Randal May, Naushad Ali, Sripathi Sureban, Eddie Bannerman-Menson, Michael Schlosser, Courtney Houchen. Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2493.

Abstract 2889: Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail

Abstract The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular mechanisms underlying the translational activation for cancer metastatic progression remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer snail followed by repression of E-cadherin expression and promotion of cell migration and invasion as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates snail expression and inhibits cell migratory/invasive capacities and metastasis. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the eIF4F translation initiation also profoundly attenuates snail translation/expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings reveal a novel role of 4E-BP1 in the regulation of EMT and metastasis through translational control of snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking snail-mediated metastatic potential of cancer. Citation Format: Qing Ye, Weijia Cai, Qing-Bai She. Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2889.

Abstract 4079: IL-6 promotes radiation-induced macrophage infiltration to tumors via upregulation of CCL2 and CCL5

Abstract Background: Radiotherapy is a major treatment modality for non-small cell lung cancer (NSCLC), and it is controversial if radiotherapy contributes to metastasis. We hypothesize that radiotherapy increases lung cancer cell recruitment of macrophage-infiltration, and the infiltrated macrophages may accelerate the metastatic potential of cancer. We examined the role of intracellular IL-6 in NSCLC cells in promoting the recruitment of macrophages after irradiation and explored the molecular mechanisms that govern the increase of macrophages. Methods: By utilizing the IL-6 knocked down (IL-6si) and scramble control (sc) A549 and H157 NSCLC cell lines, we investigated whether the intracellular IL-6 level affected macrophage migration into cancer cells. We also developed orthotopic human xenograft models in nude mice using the luciferase tagged-H157IL-6si and H157sc cells and investigated IL-6 effects on macrophage infiltration to tumor sites after irradiation. Tumors were irradiated and the irradiated mice were divided into two groups: (1) 1st group, the endogenous macrophage-infiltration into tumors was examined; and (2) in the 2nd group, mice were injected with the GFP-labeled THP-1 cells through tail veins after irradiation, and the recruitment of exogenous macrophages (THP-1) to tumor site was investigated 3 days after the injection. We also studied the molecular mechanisms by which IL-6 mediated the increase of macrophage-infiltration upon irradiation. Results: We found a decrease of macrophage recruitment by A549/H157 cells upon radiation when IL-6 was knocked down. Consistent with the in vitro results, we observed the contribution of IL-6 signaling to promote macrophage-infiltration after irradiation in vivo in human tumor xenografts. In the mechanism dissection studies, we found that CCL2 and CCL5 molecules were the critical IL-6 downstream target molecules, which mediated the radiation-induced increase of macrophage infiltration. We confirmed the contribution of these molecules by demonstrating the effects of neutralizing antibodies to these target molecules in reducing the radiation-induced enhancement of macrophage migration to tumors. Conclusion and Impacts: Study results suggest that IL-6 signaling, through the up-regulation of CCL2/CCL5, mediated the increase of macrophage infiltration to NSCLC cells after irradiation. Molecular studies suggest that blocking CCL2/CCL5 pathways can potentially be applied to conventional radiotherapy to intercept the process of radiation-promoted macrophage-infiltration. Citation Format: Xin Wang, Ying Tsai, Kwang-Hsiang Chuang, Peter Keng, Soo Ok Lee, Yuhchyau Chen. IL-6 promotes radiation-induced macrophage infiltration to tumors via upregulation of CCL2 and CCL5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4079.

460 - Investigation of the possible relationship of cancer stem cell subpopulation and metastatic potential of an oral squamous cell carcinoma cell line

460 - Investigation of the possible relationship of cancer stem cell subpopulation and metastatic potential of an oral squamous cell carcinoma cell line

Tumor stem cells from glioblastoma multiforme

Glioblastoma multiforme, a World Health Organization grade IV malignant glioma, is the most common and lethal primary brain tumor with the median survival of approximately 15–25 months after treatment. Glioblastoma multiforme has been shown to be resistant to radiotherapy and chemotherapy and invariably recurs following surgical resection and chemoradiation. The characteristics of this tumor are exemplified by heterogeneous cell population with diverse biologic properties and genetic changes, the ability to form cancer stem cells (CSC) and divided into four molecular subtypes – proneural, neural, classical and mesenchymal. Despite some success, the mechanisms leading to the formation of the most malignant tumor subtype are unclear. The aim of this review was a synthesis of modern information about the role and biological characteristics of tumor stem cells in tumor progression and the pathogenesis of glioblastoma multiforme. CSCs reside in niches, which are anatomically distinct regions within the tumor microenvironment. These niches maintain the principle properties of CSCs, preserve their phenotypic plasticity, adhesion, survival, resistance to standard cancer treatment and metastatic potential. The presence of aberrant signaling pathways (Notch, Hedgehog-Gli, Wnt/β-catenin, TGF-β/SMAD, PI3K/Akt/mTOR), both in the tumor and in the population of CSC, the dysregulation of microRNAs (miR-21, miR-128, miR-326, miR-34a), influence of epithelial-to-mesenchymal transition explains the availability of typical biological characteristics of the CSC. One needs to consider the influence of the therapy on normal stem cells in the development of drugs directed against the CSC. Regulatory mechanisms and markers found over the last decade can be used as the basis for creation of the new drugs with targeted action in the treatment of glioblastoma multiforme.

Axl Receptor Axis: A New Therapeutic Target in Lung Cancer

Abstract The Axl receptor tyrosine kinase belongs to the TAM (Tyro3, Axl, Mer) family of proteins and is upregulated in multiple types of cancers, including non-small cell lung cancer. In normal tissues, TAM receptor tyrosine kinases contribute to immune regulation. In cancer, Axl expression is associated with poor outcomes and is controlled through various transcriptional and epigenetic mechanisms. Preclinical studies have shown that Axl is involved in cancer cell migration, growth, survival and resistance to chemotherapy through activation of multiple downstream signaling pathways, such as Ras/MAPK, PI3K and Rac1. Axl is also expressed on endothelial cells and plays a role in angiogenesis. In preclinical models, Axl inhibition decreases cancer growth and impedes lung cancer metastases. Small molecule Axl inhibitors have been developed and are currently being used as monotherapy or in combination with cytotoxic chemotherapy or anti-EGFR therapy in early clinical trials. Here, we review Axl structure, functions, regulation, and preclinical and clinical studies in lung cancer.

Volatile anaesthetics enhance the metastasis related cellular signalling including CXCR2 of ovarian cancer cells

The majority of ovarian cancer patients relapse after surgical resection. Evidence is accumulating regarding the role of surgery in disseminating cancer cells; in particular anaesthesia may have an impact on cancer re-occurrence. Here, we have investigated the metastatic potential of volatile anaesthetics isoflurane, sevoflurane and desflurane on ovarian cancer cells.Human ovarian carcinoma cells (SKOV3) were exposed to isoflurane (2%), sevoflurane (3.6%) or desflurane (10.3%) for 2 hours. Metastatic related gene expression profiles were measured using the Tumour Metastasis PCR Array and qRT-PCR. Subsequently vascular endothelial growth factor A (VEGF-A), matrix metalloproteinase 11 (MMP11), transforming growth factor beta-1 (TGF-β1) and chemokine (C-X-C motif) receptor 2 (CXCR2) proteins expression were determined using immunofluorescent staining. The migratory capacities of SK-OV3 cells were assessed with a scratch assay and the potential role of CXCR2 in mediating the effects of volatile anaesthetics on cancer cell biology were further investigated with CXCR2 knockdown by siRNA.All three volatile anaesthetics altered expression of 70 out of 81 metastasic related genes with significant increases in VEGF-A, MMP-11, CXCR2 and TGF-β genes and protein expression with a magnitude order of desflurane (greatest), sevoflurane and isoflurane. Scratch analysis revealed that exposure to these anesthetics increased migration, which was abolished by CXCR2 knockdown.Volatile anaesthetics at clinically relevant concentrations have strong effects on cancer cell biology which in turn could enhance ovarian cancer metastatic potential. This work raises the urgency for further in vivo studies and clinical trials before any conclusions can be made in term of the alteration of clinical practice.

High expression of Fatty Acid-Binding Protein 5 promotes cell growth and metastatic potential of colorectal cancer cells.

Fatty acid‐binding proteins (FABPs) are responsible for binding and storing hydrophobic ligands such as long‐chain fatty acids, and for transporting these ligands to the appropriate compartments within the cell. The present study demonstrates that the FABP5 gene is upregulated in colorectal cancer cells compared to normal colon cells in a manner that correlates with disease stage and that FABP5 significantly promotes colorectal cancer cell growth and metastatic potential. Thus, FABP5 might be a promising prognostic or therapeutic biomarker candidate in human colorectal cancer.