Metastatic Phenotype Of Cancer Cells Research Articles

Mediterranean Diet Food Components as Possible Adjuvant Therapies to Counteract Breast and Prostate Cancer Progression to Bone Metastasis.

Bone metastasis is a serious and often lethal complication of particularly frequent carcinomas, such as breast and prostate cancers, which not only reduces survival but also worsens the patients’ quality of life. Therefore, it is important to find new and/or additional therapeutic possibilities that can counteract the colonization of bone tissue. High adherence to the Mediterranean diet (MD) is effective in the prevention of cancer and improves cancer patients’ health, thus, here, we considered its impact on bone metastasis. We highlighted some molecular events relevant for the development of a metastatic phenotype in cancer cells and the alterations of physiological bone remodeling, which occur during skeleton colonization. We then considered those natural compounds present in MD foods with a recognized role to inhibit or reverse the metastatic process both in in vivo and in vitro systems, and we reported the identified mechanisms of action. The knowledge of this bioactivity by the dietary components of the MD, together with its wide access to all people, could help not only to maintain healthy status but also to improve the quality of life of patients with bone metastases.

1167P DIO2 is implicated in the antitumor effect of the lung embryonic stem cell conditioned medium and impacts prognosis in non-small cell lung cancer

Critical pathways are shared between embryogenesis and cancer; but while the first is an organized and well-regulated process, the last is characterized by entropy. Several works have evaluated the potential of the embryonic microenvironment for controlling tumor development, showing that embryonic stem cell conditioned media are able to reduce metastatic phenotype of cancer cells. In this context, we have previously observed that human embryonic mesenchymal lung cells-conditioned medium (hEML-CM) are able to induce differentiation in tumor cells.

Anti-tubulin agent vinorelbine inhibits metastasis of cancer cells by regulating epithelial-mesenchymal transition

Cancer invasion and metastasis are the leading causes of death. The process of metastasis or tumor cell dissemination is still much of a mystery. Emerging evidence has shown that epithelial-mesenchymal transition (EMT) plays a vital role in the progression of malignant tumor including the inducing cell invasion and metastasis as well as promoting drug resistance. Vinorelbine is a traditional chemotherapeutic agent for treatment of lung cancer and breast cancer by the selectivity to mitotic microtubules. The aim of this study was to investigate the effect of vinorelbine on three metastatic cancer cells including lung cancer (H1975), liver cancer (HepG2), and colon cancer (HCT116) cells through inhibition of metastatic abilities and EMT program. Vinorelbine inhibited the cancer cell proliferation by MTT and colony formation assays and inducing G2/M arrest and cell apoptosis via regulation of Bax, Bcl-2, and Bcl-xL. Vinorelbine decrease the migration and invasion ability of the cancer cells by wound healing assay and Tran swell test. The molecular mechanisms of vinorelbine suppressing the metastatic phenotypes of cancer cells through modulation of E-cadherin, N-cadherin, vimentin and transcription factors Snail, MMP-2 and MMP-9. Our results demonstrated that vinorelbine inhibited the cancer cell metastasis through a reduction in metastatic mobility, such as migration, invasion, and the EMT. It provided the evidence that vinorelbine can be used alone or with other agents for treatment of metastatic lung cancer, liver cancer and colon cancer.

Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

BackgroundCarbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells.MethodsWe generated a non-shed CA IX mutant by deletion of amino acids 393–402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice.ResultsWe demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs.ConclusionsOur results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

Influence of urokinase gene-knockout in C57BL/6-PlautmI. IBugThisPlau6FDhu/GFDhu mice on growth factors in malignant melanoma

Purpose of the study. Studying characteristics of the growth factor dynamics in the intact skin, tumors and perifocal tissues of melanoma in urokinase (uPA) gene-knockout mice.Materials and methods. The study included male and female С57 ВL/6 mice (n=47) and C57BL/6‑Plautm1.1BugThisPlauGFDhu/GFDhu mice with uPA gene-knockout (n=31). В16/F10 melanoma was transplanted subcutaneously at a dose of 0.5 mL (1:10 in normal saline). Intact mice of the same strain served as controls. Levels of VEGFA, VEGFC, sVEGFR1, sVEGFR3, IGF1, IGF2, TGFβ1 and FGF21 were determined by ELISA in the skin, tumor and perifocal tissues isolated on the 21st day of the tumor growth.Results. uPA gene-knockout inhibited the growth (mostly in females) and metastasis (predominantly in males) of melanoma in mice. Inhibition of the migration of malignant cells in males could be due to low levels of TGF-β1 compared to С57 ВL/6 mice: in the skin – by 5.0 times, in tumors – by 1.8 times and in perifocal tissues – by 6.1 times. In uPA gene-knockout females, lower levels of TGF-β1 were observed in tumors – by 1.4 times inhibited metastasis, but not completely, and solitary metastatic foci were registered in the lungs. Нigh levels of IGF1 in tissues of all uPA gene-knockout mice (males: in tumors by 1.4 times, in perifocal tissues by 2.6 times, in the skin by 3.6 times; females: in tumors by 2.6 times, in perifocal tissues by 25.0 times, in the skin by 13.9 times, compared to С57 ВL/6 mice) could maintain the metastatic phenotype of cancer cells (in females) or hiher proliferative activity of melanoma cells (in males). Lower levels of FGF‑21 in tumors (males – by 5.3 times, females – by 18.4 times), perifocal tissues (males – by 9.6 times, females – by 8,5 times) and skin (males – by 6.7 times, females – by 3.3 times) in uPA gene-knockout animals could be due to the IGF‑1 growth, as their reciprocal interaction is known. Interestingly, a significant, although lesser than in mice with a normal genotype, accumulation of VEGFA in melanoma tissues was observed: in males – in tumors by 44.9 times, in perifocal tissues by 6.8 times, in the skin by 2.4 times; in females – in tumors by 5.6 times, in perifocal tissues by 2.6 times, in the skin by 3.3 times, compared to the corresponding intact controls, due to the probable involvement of the uPA receptor (uPAR) in the implementation of VEGF-induced processes.Conclusion. Changing the activity of a system of some growth factors, uPA gene-knockout modifies melanoma metabolism by inhibiting its growth and eliminating or reducing its metastatic activity.

Identification of a CIP4 PKA phosphorylation site involved in the regulation of cancer cell invasiveness and metastasis.

CDC42 interacting protein 4 (CIP4) is a CDC42 effector that coordinates membrane deformation and actin polymerization. The correlation of CIP4 overexpression with metastatic capacity has been characterized in several types of cancer. However, little information exists on how CIP4 function is regulated. CIP4 interacts with A-kinase (PKA) anchoring protein 350 (AKAP350) and CIP4 is also a PKA substrate. Here, we identified CIP4 T225 as the major CIP4 PKA phosphorylation site. In vitro and in vivo experiments using hepatocellular carcinoma (HCC) and breast cancer cells showed that expression of a CIP4(T225E) phosphomimetic mutant increased cancer cell metastatic capacity and that, conversely, expression of a CIP4(T225A) non-phosphorylatable mutant reduced invasive properties. PKA inhibition decreased to CIP4(T225A) cell-levels control but not CIP4(T225E) cell migratory and invasive efficiency. Concomitantly, our studies indicate that CIP4 T225 phosphorylation promotes the formation of functional invadopodia and enhances CIP4 localization at these structures. Our findings further provide mechanistic data indicating that CIP4 T225 phosphorylation facilitates CIP4 interaction with CDC42. Altogether this study identifies a signaling pathway that involves CIP4 phosphorylation by PKA during the acquisition of a metastatic phenotype in cancer cells.

Abstract 2849: Axl: A promising therapeutic target that leads to multimodal inhibition of the metastatic pathway

Abstract Cancer is the second leading cause of death in the United States and approximately 90% of cancer-related deaths result from metastasis, the spread of cancer cells to secondary sites. In tumors, the upregulation of certain signaling pathways promotes the metastatic phenotypes of cancer cells characterized by enhanced invasion, migration, survival, proliferation and induction of angiogenesis. A key pathway involved in metastasis is the receptor tyrosine kinase Axl. Axl is expressed in a variety of tumor types and is associated with poor prognosis, metastasis, and outcome. The purpose of the current study was to evaluate the efficacy of genetic and pharmacologic inhibitions of Axl on the metastatic phenotypes, specifically cell migration, invasion and angiogenesis. Human breast and prostate cancer cell lines (MDA-MB-231 and PC3-ML, respectively), and human lung microvascular endothelial cell (HMVEC-L) were used in this study. Stable Axl short hairpin RNA (shRNA) knockdown MDA-MB-231 and PC3-ML cell lines were generated by Mission Lentiviral transduction particles (Sigma). The efficacy of genetic and pharmacologic inhibition of Axl on tumor cell migration and invasion was evaluated by transwell migration and invasion assays. The effect of Axl knockdown on prostate cancer cell metastasis in vivo was evaluated by an intracardiac bone metastasis model. Conditioned media of Axl knockdown or control MDA-MB-231 cells were analyzed for angiogenic factors using the human angiogenesis array. The effect of Axl knockdown conditioned medium on angiogenic properties in vitro were assessed by endothelial cell tube formation and sprouting. Induction of angiogenesis in vivo was measured by performing intradermal assay. Genetic and pharmacologic inhibition of Axl decreases tumor cell migration and invasion. Axl knockdown inhibits metastatic disease burden of prostate cancer. Axl knockdown conditioned medium secretes less pro-angiogenic factors compared to its scramble control cells, suggesting that tumor cells utilize Axl pathway to promote the induction of angiogenesis. Axl knockdown conditioned medium impairs angiogenic properties, including endothelial cell tube formation and sprouting. Axl knockdown of tumor cells suppress tumor cell-induced angiogenesis in vivo. Collectively, these data indicate that Axl is involved in multiple steps of the metastatic pathway and that Axl is a promising anti-metastatic agent to inhibit tumor cell dissemination and initiation of angiogenesis. Further studies are ongoing to understand the downstream molecular signaling of Axl. Citation Format: Mai Tanaka, Dietmar W. Siemann. Axl: A promising therapeutic target that leads to multimodal inhibition of the metastatic pathway [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 2849.

Cross-Talk between Receptor Tyrosine Kinases AXL and ERBB3 Regulates Invadopodia Formation in Melanoma Cells.

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified a series of regulators, the knockdown of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia formation and function. Notably, the receptor tyrosine kinase AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. This apparent contradiction was attributed to the capacity of AXL to directly stimulate invadopodia, yet its suppression upregulates the ERBB3 signaling pathway, which can also activate core invadopodia regulators and enhance invadopodia function. Bioinformatic analysis of multiple melanoma cell lines points to an inverse expression pattern of AXL and ERBB3. High expression of AXL in melanoma cells is associated with high expression of invadopodia components and an invasive phenotype. These results provide new insights into the complexity of metastasis-promoting mechanisms and suggest that targeting of multiple invadopodia signaling networks may serve as a potential anti-invasion therapy in melanoma. SIGNIFICANCE: These findings uncover a unique interplay between AXL and ERBB3 in invadopodia regulation that points to the need for combined therapy in order to prevent invadopodia-mediated metastasis in melanoma.

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci

BackgroundThe mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality.ResultsWe identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other’s expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers.ConclusionsThis work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

Nuclear functions of NME proteins

The NME family of proteins is composed of 10 isoforms, designated NME1-10, which are diverse in their enzymatic activities and patterns of subcellular localization. Each contains a conserved domain associated with a nucleoside diphosphate kinase (NDPK) function, although not all are catalytically active. Several of the NME isoforms (NME1, NME5, NME7, and NME8) also exhibit a 3′–5′ exonuclease activity, suggesting roles in DNA proofreading and repair. NME1 and NME2 have been shown to translocate to the nucleus, although they lack a canonical nuclear localization signal. Binding of NME1 and NME2 to DNA does not appear to be sequence-specific in a strict sense, but instead is directed to single-stranded regions and/or other non-B-form structures. NME1 and NME2 have been identified as potential canonical transcription factors that regulate gene transcription through their DNA-binding activities. Indeed, the NME1 and NME2 isoforms have been shown to regulate gene expression programs in a number of cellular settings, and this regulatory function has been proposed to underlie their well-recognized ability to suppress the metastatic phenotype of cancer cells. Moreover, NME1 and, more recently, NME3, have been implicated in repair of both single- and double-stranded breaks in DNA. This suggests that reduced expression of NME proteins could contribute to the genomic instability that drives cancer progression. Clearly, a better understanding of the nuclear functions of NME1 and possibly other NME isoforms could provide critical insights into mechanisms underlying malignant progression in cancer. Indeed, clinical data indicate that the subcellular localization of NME1 may be an important prognostic marker in some cancers. This review summarizes putative functions of nuclear NME proteins in DNA binding, transcription, and DNA damage repair, and highlights their possible roles in cancer progression.

Tumor Hypoxia: A Key Determinant of Microenvironment Hostility and a Major Checkpoint during the Antitumor Response.

Recent antitumor immunotherapies such as monoclonal antibodies targeting immune checkpoints have led to outstanding results in several cancers. However, despite the favorable outcomes for responding patients, the response rate remains relatively low. This is in part due to the influence of the tumor microenvironment (TME) in protecting the tumor from the antitumor immune response and facilitating immune escape. Tumor hypoxia is one of the most important features of the TME, exerting an adverse effect on tumor aggressiveness and patient prognosis. Hypoxic stress interferes with immune plasticity and promotes tumor heterogeneity and progression. Cellular adaptation to hypoxia is primarily mediated by a family of transcriptional regulators, hypoxia-inducible factor (HIF). Apart from hypoxia, the HIF pathway is modulated in a hypoxia-independent manner. HIF-1α stabilization and activity are regulated by epigenetic changes and mutations. Strong evidence indicates that tumor hypoxia controls malignant and metastatic phenotype of cancer cells and therefore presents a unique therapeutic challenge in the treatment of solid malignancies. An alluring alternative strategy to reinvigorate anticancer immune responses comes from the emerging field of TME and its associated pathways. Targeting hypoxia or its associated pathways may therefore offer new options in the design of innovative cancer immunotherapy approaches. In this article, we briefly review the potential of hypoxic stress on tumor plasticity and stromal reactivity as well as the possible targeting of hypoxia-induced pathways to increase immunotherapy efficiency.

HCMDB: the human cancer metastasis database

Metastasis is the main event leading to death in cancer patients. Over the past decade, high-throughput technologies have provided genome-wide view of transcriptomic changes associated with cancer metastases. Many microarray and RNA sequencing studies have addressed metastases-related expression patterns in various types of cancer, and the number of relevant works continues to increase rapidly. These works have characterized genes that orchestrate the metastatic phenotype of cancer cells. However, these expression data have been deposited in various repositories, and efficiently analyzing these data is still difficult because of the lack of an integrated data mining platform. To facilitate the in-depth analyses of transcriptome data on metastasis, it is quite important to make a comprehensive integration of these metastases-related expression data. Here, we presented a database, HCMDB (the human cancer metastasis database, http://hcmdb.i-sanger.com/index), which is freely accessible to the research community query cross-platform transcriptome data on metastases. HCMDB is developed and maintained as a useful resource for building the systems-biology understanding of metastasis.

The Rac GTPase in Cancer: From Old Concepts to New Paradigms.

Rho family GTPases are critical regulators of cellular functions that play important roles in cancer progression. Aberrant activity of Rho small G-proteins, particularly Rac1 and their regulators, is a hallmark of cancer and contributes to the tumorigenic and metastatic phenotypes of cancer cells. This review examines the multiple mechanisms leading to Rac1 hyperactivation, particularly focusing on emerging paradigms that involve gain-of-function mutations in Rac and guanine nucleotide exchange factors, defects in Rac1 degradation, and mislocalization of Rac signaling components. The unexpected pro-oncogenic functions of Rac GTPase-activating proteins also challenged the dogma that these negative Rac regulators solely act as tumor suppressors. The potential contribution of Rac hyperactivation to resistance to anticancer agents, including targeted therapies, as well as to the suppression of antitumor immune response, highlights the critical need to develop therapeutic strategies to target the Rac pathway in a clinical setting. Cancer Res; 77(20); 5445-51. ©2017 AACR.

NRP2 transcriptionally regulates its downstream effector WDFY1.

Neuropilins (NRPs) are cell surface glycoproteins that often act as co-receptors for plexins and VEGF family receptors. Neuropilin-2 (NRP2), a family member of NRPs, was shown to regulate autophagy and endocytic trafficking in cancer cells, a function distinctly different from its role as a co-receptor. WD Repeat and FYVE domain containing 1 (WDFY1)–protein acts downstream of NRP2 for this function. Our results indicated that NRP2 maintains an optimum concentration of WDFY1 by negatively regulating its expression. Since increased expression of WDFY1 reduces the endocytic activity, maintenance of WDFY1 level is crucial in metastatic cancer cells to sustain high endocytic activity, essential for promotion of oncogenic activation and cancer cell survival. Here, we have delineated the underlying molecular mechanism of WDFY1 synthesis by NRP2. Our results indicated that NRP2 inhibits WDFY1 transcription by preventing the nuclear localization of a transcription factor, Fetal ALZ50-reactive clone 1 (FAC1). Our finding is novel as transcriptional regulation of a gene by NRP2 axis has not been reported previously. Regulation of WDFY1 transcription by NRP2 axis is a critical event in maintaining metastatic phenotype in cancer cells. Thus, inhibiting NRP2 or hyper-activating WDFY1 can be an effective strategy to induce cell death in metastatic cancer.

Relationship between gene mutation and lung cancer metastasis.

Cancer is a genetic disease occurring through a multi-step process. Many important genes responsible for the genesis of various cancers have been discovered, their mutations precisely identified and the pathways through which they act characterized. One question that remains unanswered is whether the development of new, more specific therapeutic agents is the best way to minimize cancer morbidity and mortality in the long-term. Metastasis is the relentless pursuit of cancer to escape its primary site and colonize distant organs. Phenotypic changes during cancer progression reflect the sequential accumulation of genetic alterations, which endow cancer cells with the ability to undergo their own divergent evolution and create distinct metastatic species. In order to understand this process, it is crucial to identify genes whose alterations accumulate during cancer progression and correlate with metastatic phenotypes of cancer cells.

Abstract 2702: WAVE3 regulates NFκB signaling and sensitizes cancer cells to apoptosis and cell death driven by TNFα.

Abstract ABSTRACT WAVE3, a member of actin nucleation proteins promotes cancer invasion and metastasis, in part through regulation of expression and activity of key matrix metalloproteinases (MMPs) MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potentially important role of WAVE3 in this pathway. Mechanistically, we found that loss of WAVE3 in cancer cells leads to significant inhibition of TNFα-induced NFκB activation as a result of de-stabilization of the IκBα-NFκB complex. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity by stabilizing the IκBα-NFκB complex. We used both pharmacological and genetic manipulations of NFκB effector molecules and identified IKKβ but not IKKα as the primary upstream target for WAVE3-mediated regulation of NFκB activity. We also showed that loss of WAVE3 resulted in a significant inhibition of TNFα-mediated MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to TNFα-driven apoptosis and cell death. Finally, we showed that either the basic rich (BR) or the proline rich (PR) domain of WAVE3 is sufficient to modulate NFkB signaling. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the stabilization of NFκB complex that promotes the metastatic phenotype of cancer cells. Therefore, therapeutic targeting of WAVE3 may prove beneficial for preventing or reducing the metastatic burden in cancer patients. Citation Format: Gangarao Davuluri, Khalid Sossey-Alaoui, Edward Plow. WAVE3 regulates NFκB signaling and sensitizes cancer cells to apoptosis and cell death driven by TNFα. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2702. doi:10.1158/1538-7445.AM2013-2702

Abstract 2816: Oncostatin M is a proinflammatory cytokine capable of exerting long term biological effects via its binding to ECM components in a bioactive conformation.

Abstract Oncostatin M (OSM) is a proinflammatory cytokine that has been reported to promote a metastatic phenotype in cancer cells via its effects on cell proliferation, attachment, migration, invasion and the epithelial to mesenchymal transition. Full length OSM is expressed as a 26 kDa MW species that can be proteolytically processed into a 22 kDa truncated species, via removal of a hydrophilic C-terminal peptide. The extracellular matrix (ECM) acts as a reservoir of biologically active molecules and as a storage site for cell signaling molecules. Many interactions between ECM proteins and cytokines are highly specific and the binding of cytokines to ECM is reported to regulate their distribution, activation and presentation to cells. In the current study, the ability of full length versus truncated OSM to bind to ECM molecules was examined, as well as the effects of immobilized OSM on T-47D and ZR-75-1 breast carcinoma cell signaling and morphology. Both full length and truncated OSM were observed to bind to the ECM components collagen-1, collagen-11, laminin and fibronectin. At least two distinct binding sites were apparent for OSM binding to ECM proteins. Full length 26 kDa OSM was observed to bind ECM to a greater extent at neutral pH than truncated 22 kDa OSM, which indicated a role for the C-terminal region of OSM in ECM binding. Under acidic conditions (pH 5.5) similar amounts of full length versus truncated OSM bound to ECM, which indicated binding independent of the C-terminal tail of OSM. Electrostatic interactions appeared to mediate OSM binding to ECM, since greater binding of 26 kDa and 22 kDa to ECM was observed at pH 5.5 versus pH 7.5 and a slow detachment of OSM bound to ECM at pH 5.5 was observed when the pH was raised to neutral. OSM bound to ECM was observed to be protected from cleavage by tumor-associated proteases, when compared to proteolysis of unbound OSM, which suggested a stabilizing effect on OSM. Importantly, OSM bound to ECM was demonstrated to be biologically active and able to induce pSTAT3 and other OSM associated proteins in T-47D and ZR-75-1 breast carcinoma cells, in vitro. The induction of pSTAT3 could be inhibited with an OSM neutralizing antibody indicating that the ECM binding sites were distinct from the OSM receptor binding site. In addition, OSM bound to ECM was observed to induce morphological changes in T-47D breast cancer cells that included the formation of invadapodia. Taken together, this data suggests an important role for OSM bound to ECM in tumor metastasis, as well as in the acquisition of chronic inflammation and provides additional evidence for the role of inflammatory processes in cancer. Citation Format: Randall E. Ryan, Bryan Martin, Liliana Mellor, Owen McDougal, Julia Oxford, Cheryl Jorcyk. Oncostatin M is a proinflammatory cytokine capable of exerting long term biological effects via its binding to ECM components in a bioactive conformation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2816. doi:10.1158/1538-7445.AM2013-2816

Abstract 2704: Ectopic expression of CHST11 promotes proliferation and migration of human breast cancer cells.

Abstract Introduction. We have previously demonstrated that the expression levels of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness (basal-like, triple-negative and mesenchymal phenotype) of human breast cancer. The present study was performed to establish stably transfected cell lines and study the functional role of CHST11 in human breast cancer cells. Methods. MCF7 cells were transfected with a plasmid vector containing the CHST11 coding sequence. Stable transfectants, MCF-7-CHST11, were established. Flow cytometry and real-time PCR were used for expression analysis. Metastatic phenotype of cells was characterized through conduction of proliferation and migration assays. Morphology and growth pattern of MCF-7-CHST11 cells were monitored and compared to parental MCF-7 cells. Results. We observed that MCF-7-CHST11 reacted strongly with anti-chondroitin sulfate A (CS-A) antibody and recombinant P-selectin. CS-A is the immediate product of CHST11 activity. CHST11 expression significantly affected cell proliferation as MCF-7-CHST11 cells grew much faster. Migration assay using transwell chamber showed high migratory potential for MCF-7-CHST11 cells as compared to the parental MCF-7 cells. The significant migratory advantage was not proliferation dependent. The CHST11expression affected the morphology of cells and cell spreading behavior consistent with more efficient anchorage independent growth. Colony formation in soft agar, Invasion and adhesion assays are being performed at the present time. We are planning more experiments to study mechanisms involved. Conclusions. The data confirm that the CHST11 products on the cell surface can serve as P-selectin ligands. The results indicate that the CHST11 expression can affect metastatic phenotype of cancer cells by promoting cellular proliferation and migratory potential. Citation Format: Alice Behrens, Fariba Jousheghany, Aiwei Yao-Borengasser, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Ectopic expression of CHST11 promotes proliferation and migration of human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2704. doi:10.1158/1538-7445.AM2013-2704

Abstract 273: Molecular markers promoting metastases to the brain via EMT: Genes, proteins, and functional analysis.

Abstract Brain metastasis is the most common cause of mortality and morbidity of many cancers and occurs in more than 40% of patients. The metastatic cascade entails an orderly sequence of steps enabling tumor cells to migrate from the primary tumor and colonize at secondary locations. The underlying mechanisms of metastatic dissemination to the brain remain to be defined. However, In order to achieve this metastatic potential a cancer cell may go through a cellular reprogramming process, namely epithelial-to-mesenchymal transition (EMT), and its counterpart mesenchymal-to-epithelial transition (MET), by acquiring multiple genetic alterations and a stem cell like phenotype. We hypothesized that EMT would play a crucial role in brain metastasis. In order to achieve our goal we studied the expression of markers of EMT, MET, and stem cells in metastatic brain tumor samples. Microarray analysis of metastatic tumors was also done using a plate form of 17,000 genes. Furthermore, functional analysis establishing the metastatic phenotype of cancer cells towards a cerebral environment was also demonstrated using metastatic cell lines. Our results demonstrated that the expressions of EMT markers (Snail, TWIST, pSTAT3, NFkB), as well as the mesenchymal marker Vimentin, were present in metastatic tumor samples. The stem cell marker CD44 was also highly expressed. Gene expression analysis established the high presence of transcriptional factors associated with differentiation and reprogramming. Lastly, Immunofluorescence analysis demonstrated the presence of the mesenchymal marker Vimentin in brain tumor specimens. Tumor cells grown in astrocytic media displayed an increased cell proliferation, as well as enhanced S-phase cell cycle entry using an EDU incorporation technique. Migration of primary tumor cells was significantly enhanced in astrocytic media as evidenced by scratch wound migration. Furthermore, the tumor cells have a greater affinity for astrocytic media as demonstrated by chemotatic migratory tests. Moreover, co-culture analysis of primary tumor cells with astrocytic cells shows amicable mutual growth with Immunofluorescence demonstrating cell-to-cell interaction via E-Cadherin. In conclusion results of this study suggests that reprogramming via EMT/MET plays a crucial step in brain metastasis, and that the cerebral milieu provides a suitable microenvironment for metastatic cells to grow and disseminate. Citation Format: Dhruve S. Jeevan, Sudeepta Sridhara, Alex Braun, Raj Murali, Meena Jhanwar-Uniyal. Molecular markers promoting metastases to the brain via EMT: Genes, proteins, and functional analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 273. doi:10.1158/1538-7445.AM2013-273

Impact of host ageing on the metastatic phenotype

Ageing impacts multiple host mechanisms involved in cancer progression. Here we show that poorly metastatic Lewis lung carcinoma (LLC) cells form less bulky metastatic deposits in aged mice (>52 weeks) relative to their young (4–6 weeks) counterparts. Serial selection of LLC cells for increased metastatic capability in either young or old mice led in both cases to exaggerated growth of pulmonary nodules after only 5 cycles of in vivo passage. The respective metastatic cellular variants established in young (Y-series) or old (O-series) mice differed in cell morphology and constitutive activity of growth factor receptors, especially phospho-PDGFRa and phospho-EPHA7. These cell lines also exhibited marked differences in their time dependent profiles of cellular impedance (CI), which reflects their physical properties, such as cell shape, adhesion and interactions with substrata. In confluent monolayer culture Y-series cell lines generated high and increasing CI values, while these values remained low and constant in the O-series of cell lines. These observations suggest that the selective pressure of the metastatic microenvironment in young versus old hosts is sufficiently different to results in the enrichment of distinct, age-related metastatic phenotypes of cancer cells. Thus, age could inform therapeutic approaches to metastatic cancers.