Potential Of Cancer Cells Research Articles

Abstract LB-321: TP63 isoforms regulate distinct and cooperative transcriptional signatures that drive cancer progression and predict clinical outcomes

Abstract TP63 is required to maintain stem cell pluripotency and suppresses the metastatic potential of cancer cells through multiple mechanisms. Even though roles for Tp63 in cancer have been described, the transcriptional roles of TP63 isoforms, namely TAp63 and ΔNp63, in tumor development and progression have been perplexing due to their variable expression in tissues, their complex interaction with each other and with TP53, and the lack of adequate antibodies to distinguish between their isoforms. Of concern is the use of total p63 as a diagnostic marker for many epithelial cancers in the clinic with disregard of the existence and functions of the various isoforms, which may lead to improper diagnosis. These notions necessitate a deeper understanding of how the distinct transcriptional programs controlled by TP63 isoforms affect cancer progression and patient outcomes. To shed light on the activities of the TP63 isoforms in cancer development and progression, we conducted a pan-cancer analysis of The Cancer Genome Atlas (TCGA) to identify the transcriptional networks regulated by TAp63 and ΔNp63 using transcriptomes derived from epidermal cells of TAp63-/- and ΔNp63-/- mice. We then derived 17 cancer developmental (tumor versus corresponding normal) and 27 cancer progression (high stage versus low stage) signatures. Our analysis revealed a consistent tumor suppressive pattern for TAp63. In contrast, we identified pleiotropic roles for ΔNp63 in tumor development and progression. Our findings revealed a cooperative role between TAp63 and ΔNp63 via their gene signatures that predict survival and correlates with progression of cancer patients in bladder cancer (BLCA), clear cell renal cell carcinoma (KIRC), papillary renal cell carcinoma (KIRP) and low grade glioma. Importantly, we found that ΔNp63 transcriptional activity integrates the remodeling of extracellular matrix remodeling to provide an ideal environment in these cancers to undergo epithelial to mesenchymal transition and gain pluripotent stem cell characteristics, thus supporting tumor progression, while TAp63 activity acts to regulate the cell cycle required in stem cell maintenance which may further allow tumors to progress. These signatures are not only predictive of survival across stages, but can even stratify patients within the same stage into different survival groups in the genitourinary tumors as well as in low grade gliomas. We then validated our findings in independent cohorts. Our data describe a global approach for understanding transcriptional activities of TP63 isoforms across a large number of cancer types, potentially enabling the identification of patient subsets most likely to benefit from therapies predicated on manipulating specific TP63 isoforms in order to inhibit the acquisition of pluripotency potential. Citation Format: Hussein Abbas, Kimal Rajapakshe, Justin Wong, Ngoc Hoang Bao Bui, Preethi Gunaratne, Kenneth Tsai, Cristian Coarfa, Elsa Flores. TP63 isoforms regulate distinct and cooperative transcriptional signatures that drive cancer progression and predict clinical outcomes. [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 LB-321.

Abstract 4572: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis

Abstract BACKGROUND: The binding of the homeostatic chemokine SDF-1 to the G -protein-coupled receptor C-X-C Chemokine Receptor 4 (CXCR4) induces cell migration via heterotrimeric G-proteins of the Gi family. Additionally, activated CXCR4 can stimulate the small GTPase RhoA through Gα13 which is required for directional cell migration, contributing to the metastatic potential of cancer cells. Recently, GPCR hetero-oligomerization has emerged as a means by which new signaling entities can be created, yet how receptor heteromers affect receptor pharmacology remains largely unknown. Here we report that agonist-bound CXCR4 is able to form a heteromer with agonist-bound cannabinoid receptor 2 (CB2) at the cell membrane of prostate cancer (PCa) cells and that receptor interaction inhibits the activation of the cytoskeleton regulator RhoA. EXPERIMENTAL METHODS: To confirm CXCR4-CB2 interactions on the cell membrane, we utilized an in situ proximity ligation assay to detect heterodimer formation on the surface PC-3 cells treated with SDF-1 alone or, simultaneous treatment with SDF-1 and the CB2 agonist AM1241. In order to establish that RhoA activity decreased upon heterodimerization of CXCR4 and CB2, we performed RhoA-GTP pulldown assays in PC-3 cells. We then tested the effects of CXCR4/CB2 heterodimerization on actin cytoskeletal reorganization by performing immunocytochemistry using an anti-Phalloidin antibody to detect F-actin. The ROCK kinase inhibitor Y-27632 was used as positive control of RhoA inhibition in these studies. Next, we assessed the effects of receptor heterodimerization on cellular adhesion to extracellular matrix components as the ability of cancer cells to adhere to the endothelium is critical of transendothelial migration during tumor cell intravasation and extravasation. Intuitively, alterations in adhesion capabilities should affect the ability of PCa cells to migrate through an endothelial cell monolayer. Thus we performed transendothelial migration assays. RESULTS: The proximity ligation assay revealed that CXCR4 and CB2 physically interacted on the cell surface of prostate cancer cells. Furthermore, receptor heterodimerization diminished SDF-1 induced actin cytoskeleton reorganization as characterized by membrane ruffling in a similar manner to the ROCK kinase inhibitor Y-27632. Heterodimer formation, stimulated by simultaneous treatment of cells with SDF-1 and AM1241 also attenuated PCa adhesion and transendothelial migration. CONCLUSION The attenuation of RhoA has multiple functional consequences leading to a less metastatic phenotype. Thus induction of CXCR4-CB2 heterodimerization is a viable pharmacological approach to targeting the CXCR4-RhoA axis as a means of preventing the metastatic dissemination of PCa cells. Citation Format: Kisha A. Scarlett, Christopher J. Coke, Imani Fennell, Cimona V. Hinton. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis. [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 4572.

Acquired resistance to HSP90 inhibitor 17-AAG and increased metastatic potential are associated with MUC1 expression in colon carcinoma cells.

Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances the survival, growth, and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colon cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. Cross-resistance was found with another HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin. The resistant cells showed obvious morphology changes with a metastatic phenotype and significant increases in migration and adhesion to collagens. Western blotting analysis of epithelial-mesenchymal transition molecular markers found that expression of E-cadherin downregulated, whereas expression of N-cadherin and β-catenin upregulated in the resistant cells. Mucin 1 (MUC1) has been reported to mediate metastasis as well as chemical resistance in many cancers. Here, we found that MUC1 expression was significantly elevated in the acquired drug resistance cells. 17-AAG treatment could decrease MUC1 more in parental cells than in acquired 17-AAG-resistant cells. Further study found that knockdown of MUC1 expression by small interfering RNA could obviously re-sensitize the resistant cells to 17-AAG treatment, and decrease the cell migration and adhesion. These were coupled with a downregulation in N-cadherin and β-catenin. The results indicate that HSP90 inhibitor therapies in colon carcinomas could generate resistance and increase metastatic potential that might mediated by upregulation of MUC1 expression. Findings from this study further our understanding of the potential clinical effects of HSP90-directed therapies in colon carcinomas.

RO-19RFQMR THERAPY IN BRAIN TUMORS IN CHILDREN AND YOUNG ADULTS

RO-19. RFQMR THERAPY IN BRAIN TUMORS IN CHILDREN AND YOUNG ADULTS G.S. Nayar1, Rajah Vijay Kumar2, Anjana Nair1, and Meena Augustus3; Any Time Medicare Services, Bengaluru, Karnataka, India; Scalenene CARD, Bengaluru, Karnataka, India; Shreis Scalene Services, Gaithersburg, MD, USA Rotational Field Quantum Magnetic Resonance (RFQMRw) Therapy (United States Patent 2007/ 0208249 A1 (19) Sep. 6, 2007) has evolved from the Magnetic Resonance Imaging technology. Highly complex electromagnetic beams in the radio frequency spectrum are precisely controlled and focused on to tissues to alter the cell membrane potential of the target tissue cells which in turn stimulates tissue growth in degenerative diseases such as Osteoarthritis with increase in cartilage thickness or triggers apoptosis (programmed cell death) and stops the growth in solid malignant tumors of tissues and organs like brain, liver, lung and others. Cell membrane potentials are created by the difference in the concentration of ions inside and outside the cell creating an electrochemical force across the membrane. Cell membrane potential helps control cell membrane permeability and downstream cellular pathways that can be “controlled”. When cancerous, sodium and water flow into the cells and K, Mg, Ca and Zn are lost from the cell interior and cell membrane potential falls. This phenomenon is altered by RFQMR, which “engineers” the membrane potential of cancer cells and induces cell death or apoptosis. The medical device to deliver RFQMR therapy, Cytotronw, is being used effectively in India and a few other countries, both for tissue regeneration and tumor destruction. The technology and protocol in management of cancer are briefly discussed with a few cases of advanced brain tumors especially in the young where safety and absence of side effects are highlighted. A few cases are discussed. Neuro-Oncology 18:iii159–iii164, 2016. doi:10.1093/neuonc/now082.19 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Review on near-infrared heptamethine cyanine dyes as theranostic agents for tumor imaging, targeting, and photodynamic therapy.

A class of near-infrared fluorescence (NIRF) heptamethine cyanine dyes that are taken up and accumulated specifically in cancer cells without chemical conjugation have recently emerged as promising tools for tumor imaging and targeting. In addition to their fluorescence and nuclear imaging-based tumor-imaging properties, these dyes can be developed as drug carriers to safely deliver chemotherapy drugs to tumors. They can also be used as effective agents for photodynamic therapy with remarkable tumoricidal activity via photodependent cytotoxic activity. The preferential uptake of dyes into cancer but not normal cells is co-operatively mediated by the prevailing activation of a group of organic anion-transporting polypeptides on cancer cell membranes, as well as tumor hypoxia and increased mitochondrial membrane potential in cancer cells. Such mechanistic explorations have greatly advanced the current application and future development of NIRF dyes and their derivatives as anticancer theranostic agents. This review summarizes current knowledge and emerging advances in NIRF dyes, including molecular characterization, photophysical properties, multimodal development and uptake mechanisms, and their growing potential for preclinical and clinical use.

A homeopathic nosode, Hepatitis C 30 demonstrates anticancer effect against liver cancer cells in vitro by modulating telomerase and topoisomerase II activities as also by promoting apoptosis via intrinsic mitochondrial pathway

ObjectiveHomeopathic nosodes have seldom been scientifically validated for their anticancer effects. This study was conducted to examine if a recently developed hepatitis C nosode has demonstrable anticancer potential in cancer cells in vitro. MethodsAnticancer effects of Hepatitis C 30C (Hep C 30), if any, were initially tested on three cancer cell lines, HepG2 (liver cancer), MCF-7 (breast cancer) and A549 (lung cancer) and one normal liver cell line WRL-68 cells and subsequently a more thorough study using further scientific protocols was undertaken on HepG2 cells (against WRL-68 cells as the normal control) as HepG2 cells showed better anticancer response than the other two. Three doses, one at 50% lethal dose (LD50) and the other two below LD50, were used on HepG2 cells subsequently. Protocols like apoptosis induction and its possible signaling mechanism were deployed using immunoblots of relevant signal proteins and confocal microscopy, with particular reference to telomerase and topoisomerase II (Top II) activities, two strong cancer biomarkers for their direct relationship with divisional activities of cells and DNAs. ResultsHep C 30 induced apoptosis, caused distorted cell morphology typical of apoptotic cells, increased reactive oxygen species generation and produced increased DNA nicks. Further it enhanced pro-apototic signal proteins like Bax, cytochrome c and inhibited anti-apoptotic signal proteins, Bcl-2, cytochrome c and caspase-3, changed mitochondrial membrane potential and caused externalization of phosphatidylserine. The drug also decreased expression of two cancer biomarkers, Top II and telomerase, consistent with its anticancer effect. ConclusionHep C 30 has demonstrable anticancer effects against liver cancer cells in vitro.

Sequential analysis of circulating tumor cells on genome and protein level: potential regulation of the invasion marker CapG by PIK3CA

Overexpression of macrophage capping protein (CapG) results in increased invasion potential in cancer cells. PI3K driven signal-transduction could lead to an increased CapG expression. Here we show isolated single CTC characterization for the invasion marker CapG and PIK3CA mutations.

Abstract P1-03-07: The "panta rhei" of breast cancer: Gene expression timeline analysis during progression of microinvasive breast cancer microenvironment

Abstract Background. Tumors develop by progression through a series of stages. It is now widely accepted that cancer is attributed to the accumulation of genetic alterations in cells. Every cells of the tumor microenvironment is constantly changing in the flow of the cancer progression. A number of genes have been identified as having functions in various stages of progression in promoting cancer progression in experimental models. However, the association between gene expression alterations and resulting phenotypic alterations with respect to the aggressiveness and migration potential of cancer cells is not fully understood. Therefore, elucidation of genotype–phenotype correlation will be required to further understand the complex process of progression and invasion. All tumors require at least some stroma to meet their needs of nutrition, waste removal, and structure. It has become clear in recent years that stroma is essential for tumor maintenance and growth and has potential as a therapeutic target. Here, we aimed to give a chronological order of gene expression changes given in the dynamical framework of microinvasive breast cancer microenvironment. Materials and Methods. RNA-seq (Ion Proton technology) was performed on three microinvasive breast cancers, applying new modifications to the usual protocol. For each of them we microdissected 7 different portions of the tumor (around 200 cells), 4 related to the breast epithelium and 3 to the stroma. The regions were selected on the basis of their grade of progression. Breast epithelium was chronologically subdivided in normal breast epithelium (NBE), carcinoma in situ (CIS), emerging invasive fingers (EIF) and invasive breast cancer (IBC). For each of the breast epithelium subdivisions we collected the adjacent stroma (S) except for the in situ portion: S-NBE, S-EIF and S-IBC. Results: Whole transcriptome analysis performed on each microdissected regions reveals a series of gene expression changes occurring during cancer progression in the breast epithelium along with the adjacent stroma. The dendogram analysis, based on the whole gene expression data of each patient revealed a perfect group organization of the various microdissected portions of stroma and mammary epithelium. Within the dendogram, the organization of Normal, In Situ, EIF and Invasive tissue respected perfectly the biological assumptions. Conclusions: More thorough analyses are needed to give a clear view of the flow of molecular events starting from the normal breast epithelium to the microinvasive stage, as well as to give a better understanding of the stroma-epithelium molecular means of communication. The analysis of all the molecular changes occuring in the breast epithelium and in the stroma of microinvasive cancer could lead to the development of new therapeutic targets. Citation Format: Lessi F, Scatena C, Aretini P, Menicagli M, Franceschi S, Ortenzi V, La Ferla M, De Gregorio V, Bevilacqua G, Naccarato GA, Mazzanti CM. The "panta rhei" of breast cancer: Gene expression timeline analysis during progression of microinvasive breast cancer microenvironment. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-07.

Abstract 1444: Docosahexaenoic acid reduces cancer cell migration may link with actin binding proteins and miRNA 17-92 cluster expressions changes

Abstract Metastasis and progression, reflected by higher migration and longer cell survival, are major contributing factors in making cancers the leading cause of death worldwide. Cancer cells exhibit higher migration rates and greater cell viability facilitated by actin binding protein-mediated cytoskeletal remodeling and secondary messengers (cAMP and/or cGMP) signaling. Studies have shown that docosahexaenoic acid (DHA), a poly unsaturated fatty acid, inhibits cancer cell metastatic phenotype. Moreover, miRNA-17∼92 cluster has been reported to be highly expressed in metastasis. Therefore, we put forward a hypothesis that DHA will induce changes in actin binding protein dynamics and miRNA-17∼92 cluster expression to inhibit cancer cell migration and viability. Non-cance (MLE12) and cancer (A549) cells were treated with 8-Br-cAMP and/or DHA for 30 min, 3h, 6h and 24h. Actin binding proteins (profilin, cofilin, vimentin and gelsolin) and miRNA-17∼92 cluster expression were analyzed by western blot and qRT PCR respectively. Cell migration was estimated by wound assay and transwell apparatus. F-actin content and actin binding proteins were measured using confocal microscopy at the leading edges of wound. Proliferation and cell viability were estimated by flow cytometer, IF using ki-67, and cleaved-caspase-3 antibodies. F-actin content, cell migration and proliferation were increased while cell viability was decreased by cAMP. DHA treatment reverses the increase in actin content and cell migration in cancer cells but not in non-cancer cells. These findings correlate with changes in actin binding protein content and miRNA 17∼92 cluster expression. Thus, DHA specifically inhibits cancer cell migration, proliferation, and viability and this effect correlates with decreases in actin binding protein levels and miRNA-17∼92 cluster expression. In conclusion, DHA supplementation suppresses cellular changes related to metastatic potential in cancer cells and could provide therapeutic potential against cancer cell metastasis. Moreover, actin binding proteins and miRNA-17∼92 cluster expression could serve as innovative therapeutic targets and biomarkers for cancer progression. Citation Format: Mehboob Ali, Kathryn M. Heyob, Lynette K. Rogers. Docosahexaenoic acid reduces cancer cell migration may link with actin binding proteins and miRNA 17-92 cluster expressions changes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1444. doi:10.1158/1538-7445.AM2015-1444

Abstract 4989: Cannabinoid receptor 2 and C-X-C chemokine receptor 4 interact to abrogate CXCL12-mediated cellular response

Abstract The expression of C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with increased metastatic potential of cancer cells. CXCR4 increases tumor malignancy by encouraging tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis provides a good strategy to inhibit the metastatic spread of tumor cells and slow cancer progression. Recent studies suggest that cannabis may have anti-proliferative as well as anti-metastatic properties, though a biochemical mechanism describing how this occurs has yet to be discovered. Our lab has confirmed that an interaction between agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) can decrease cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231 with CXCL12 and AM1241, a synthetic ligand for CB2, desensitized the intrinsic cellular response to migrate toward areas of high CXCL12 concentration. Furthermore, through co-immunoprecipitation, we determined that there was increased interaction between the two receptors with co-stimulation. When CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Moreover, when cells were treated with the CB2 antagonist (AM630) and CXCL12 simultaneously, the decrease in migration was no longer seen, as CXCL12 was able to induce cell movement. In order to determine whether receptors were activated in response to simultaneous stimulation, calcium mobilization assays were performed and results showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treat with their individual ligands. Therefore, we propose that the interaction of CB2 with CXCR4 may play a role in inhibiting the cells response to CXCL12, leading to a loss in metastatic potential and presenting a new mechanism for regulating oncogenic signaling. Citation Format: Christopher Coke, Ahriea Johnson, Kia Jones, Cimona Hinton. Cannabinoid receptor 2 and C-X-C chemokine receptor 4 interact to abrogate CXCL12-mediated cellular response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4989. doi:10.1158/1538-7445.AM2015-4989

Abstract 4468: Anticancer activity of novel cucurbitacin analogue in pancreatic cancer

Abstract Background: Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States. Accumulating studies have witnessed the malfunction of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), enhances patient survival by only few months. Cucurbitacins, naturally occurring dietary tetracyclic triterpenoid compounds, have shown promising anti-cancer activities. Herein, we investigated the potency and anti-cancer efficacy of a novel analogue of cucurbitacin (Cuc D) in pancreatic cancer cell lines and in a xenograft mouse model. Additionally, we determined efficacy of this analogue on MicroRNAs (miRNAs) which are important regulators of genes that have crucial roles in pancreatic tumorigenesis. Methods: The effect of Cuc D on the growth of pancreatic cancer cells was determined by cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96 h. The clonogenic potential of cancer cells was also studied using the colony formation assay. Tumor suppressor miR-145, which is downregulated in pancreatic cancer, directly target MUC13. Thus the effect of Cuc D was also investigated on the expression of miR-145 through qPCR analysis. Immunoblotting techniques were performed to study the known direct targets of miRNA-145 and its associated proteins. The anti-cancer potential of Cuc D in pancreatic cancer was also evaluated in vivo using a xenograft mouse model. Results: Our results demonstrate potent anticancer effects of Cuc D on pancreatic cancer cells. Cuc D induces dose and time dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell line models at nanomolar concentrations (100-500 nM). It also inhibits colony formation and invasiveness of pancreatic cancer cells. Furthermore, Cuc D blocks the cell cycle progression in G2/M phase and decreases the mitochondrial membrane potential in pancreatic cancer cells. Notably, Cuc D significantly increases the expression of tumor suppressor miR-145 in HPAF-II cells as observed by qPCR. Furthermore, Cuc D decreases the expression of MUC13 and its associated proteins including pAKT and HER2. In addition, it restores the expression of p53 level as studied by immunofluorescence technique. The expression of key oncogenic proteins including NF-κB, STAT3 (Tyr-705) and Mcl-1 were also downregulated. The levels of PTEN and p27 (Kip1) tumor suppressor genes were increased after Cuc D treatment. Additionally, in vivo administration of Cuc D effectively inhibited pancreatic tumor growth in xenograft mouse model. Conclusion: Overall, this study suggests that Cuc D modulates the expression of key oncogenes and tumor suppressors, thus it can be a promising therapeutic modality for pancreatic cancer prevention and treatment. Citation Format: Mohammed Sikander, Sheema Khan, Neeraj Chauhan, Mohd Saif Zaman, Murali Mohan Yallapu, Fathi T. Halaweish, Bhavin Chauhan, Shabnam Malik, Meena Jaggi, Subhash C. Chauhan. Anticancer activity of novel cucurbitacin analogue in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2015-4468

Abstract 3354: Cancer-associated or cirrhosis fibroblasts recovered from hepatocellular carcinoma promote macrophages and cancer cells to progressive phenotype

Abstract Background/Aim: Tumor microenvironment plays supportive roles for cancer cells of proliferating, invading and spreading systemically. Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) are deemed to be essential constituents forming cancer-surrounding niche. However, the cross-talks between CAFs and TAMs in the settings of hepatocellular carcinoma (HCC) has yet to be deciphered. The lack of a reliable system simulating in vivo-microenvironment has been one of the obstacles hampering investigation of CAFs and TAMs. By using the newly-established model consisting of fibroblasts directly recovered from the disease liver, we aimed to clarify the mechanisms of CAFs modulating the macrophage differentiation and malignant potential of cancer cells in order to search for therapeutic targets. Methods: We enrolled three patients who underwent the resection of HCC and two who did living donor liver transplantation. From the resected or the explanted livers, fibroblasts from cancer and its adjacent tissues were separated by tissue digestion. We examined their ontogeny by the expression of fibroblast-specific markers. For the functional analyses, we examined invasiveness of HuH7 cells on matrigel and migratory response of monocytes in the presence of conditioned media (CM) from the fibroblasts. We assessed the phenotypes/function of macrophages after the differentiation from monocytes in the presence of M-CSF and the CM. We comprehensively assayed cytokine/chemokine in the CM to identify functionally-relevant factors. Results): After the culture, we established three types of fibroblasts, CAFs, cirrhotic liver fibroblasts (LCFs) and non-cancerous fibroblasts (NFs). All of these were positive for vimentin, SMA, PDGFRa and FAP (>99%), confirming that they are ontologically fibroblasts instead of being HCC, hepatocytes or biliary cells. The invasiveness of HuH7 were higher in the presence of the CAF- or LCF-CM compared to those with NF-CM. Similar superiority was observed with the CAF- or LCF-CM in the migratory ability of monocytes. In the process of macrophage differentiation, the CAF- or LCF-CM were capable of polarizing macrophages into TAM/M2 subtypes, as evidenced by their higher production of IL-10 but lesser IL-12. In the CAF- or LCM-CM, the levels of IL6, IL-8, CCL2 and GRO were higher than those in the NF-CM. Conclusion: The CAFs as well as LCFs directly recovered from the cirrhotic liver are functionally competent of driving TAM/M2 differentiation and providing malignant potential to cancer cells, thereby tuning the microenvironment favoring HCC development. Citation Format: Yohei Mano, Tatsuya Kanto, Hirotaka Shoji, Schiyo Yoshio, Masaya Sugiyama, Yosuke Osawa, Kiminori Kimura, Ken Shirabe, Yoshihiko Maehara, Masashi Mizokami. Cancer-associated or cirrhosis fibroblasts recovered from hepatocellular carcinoma promote macrophages and cancer cells to progressive phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3354. doi:10.1158/1538-7445.AM2015-3354

Abstract 4177: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased angiogenesis

Abstract Tumor angiogenesis is a complex process modulated by numerous signaling molecules and pathways that influence cell migration, invasion, survival and tube formation. The expression of the G-protein-coupled receptor (GPCR) C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with the increased angiogenic potential of cancer cells. The ligand-specific binding of CXCL12 to CXCR4 increases tumor angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF) at both mRNA and protein levels. Thus, modulating the CXCR4/CXCL12 signaling axis provides an attractive target for the inhibition of tumor angiogenesis and cancer progression. Recent studies have suggested that in addition to its palliative properties, cannabis may have anti-proliferative and anti-metastatic effects by acting through the GPCR, cannabinoid receptor 2 (CB2R). GPCR heterodimerization has emerged as a means by which new signaling entities can be created with respect to agonist binding. Preliminary co-immunoprecipitation data from our lab indicates that agonist-bound CXCR4 and agonist-bound CB2R interact at the cell membrane and that receptor interaction mitigates the effects of CXCL12-mediated CXCR4 signaling. Here we show that co-stimulation of prostate cancer cells with CXCR4 and CB2R-specific agonists results in decreased VEGF expression in vitro. Furthermore, treatment of human endothelial cells with conditioned medium collected from co-stimulated prostate cancer cells decreased endothelial cell migration in wound healing and invasion assays, and decreased endothelial cell tubulogenesis. Taken together, these results demonstrate that desensitization of CXCR4 signaling through association with CB2R is a novel anti-angiogenic strategy and reinforces the clinical relevance of cannabinoids in cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Kisha A. Scarlett, Brittney Sandifer, Christopher J. Coke, Cimona V. Hinton. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased angiogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4177. doi:10.1158/1538-7445.AM2015-4177

Abstract 5368: Metarrestin effectively disassembles PNCs and inhibits metastasis

Abstract The perinucleolar compartment (PNC) is a nuclear body that forms specifically in highly malignant cancer cells. PNC prevalence is shown to reflect metastatic potential of cancer cells from solid tissue origins. A high-content screen was performed to identify small molecules that reduce PNC prevalence. Medicinal chemistry optimizations of confirmed hits yield metarrestin, having 95% of oral bioavailability and a PNC disassembly IC50 between 100 to 400 nM across multiple cancer cell lines. Metarrestin inhibits soft agar growth and the invasive capabilities of cancer cells in vitro. In vivo mouse xenograft metastatic studies showed that metarrestin effectively inhibits lung and/or liver metastasis of prostate cancer (PC3M), pancreatic cancer (organotypic spheroids of PANC1), and a breast cancer patient xenograft (PDX) model derived from metastatic cells isolated from a pleural effusion. Treated animals tolerate metarrestin well with no discernable impact on animal well-being including weight compared with control animals. These data supports the idea that the pharmacological disassembly of PNC produces the inhibition of malignant behavior of cancer cells both in vitro and in vivo. Cellular and molecular analyses of the mechanisms of action of metarrestin show that this compound induces significant and reversible changes in the nucleolar structure, accompanied by a selective reduction of pol I transcription. These studies suggest that PNC structural integrity directly associates with pol I function and nucleolar structure. Experiments are currently underway to identify the cellular factors and pathways by which metarrestin reduces PNCs, disrupts nucleolar structure, regulates transcriptions, and blocks malignant behaviors. Citation Format: Chen Wang, Kevin Frankowski, Teper Yaroslav, Samarjit Patnaik, Frank Schoenen, Noel Southall, Wei Sun, Steve Titus, Lesley Griner, Christopher Dextras, Jamey Sultan, Irawati Kandela, Marzena Lewandowska, Yi-Ping Wen, John Norton, Jin Sol Kang, Andrew Mazar, Wei Zhang, Jeffrey Aubé, Marc Ferrer, Udo Rudloff, Juan Jose Marugan, Sui Huang. Metarrestin effectively disassembles PNCs and inhibits metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5368. doi:10.1158/1538-7445.AM2015-5368

Abstract 4956: Hsp27 negatively affects Hippo tumor suppressor pathway to regulate cell survival in cancer

Abstract Introduction: Heat shock protein 27 (Hsp27) is a molecular chaperone highly and uniformly expressed in treatment resistant cancers like castrate resistant prostate cancer (CRPC). Hsp27 regulates activity of several oncogenic pathways and its levels correlate with aggressive tumor behaviour, drug resistance and tumor growth. Similarly, dysregulation of the Hippo tumor suppressor pathway, which restricts organ size and cell proliferation, occurs in many types of cancers. In healthy cells, activation of the Hippo pathway results in phosphorylation and cytoplasmic retention of two transcriptional co-activators YAP and TAZ, whereas in cancer YAP/TAZ are free to translocate to the nucleus and increase cell proliferation by promoting the activities of certain transcriptional factors including TEAD1. Inactivation of the Hippo pathway correlates with poor patient outcome and progression of tumors as well as an increase in migration, invasion and metastatic potential of cancer cells. Therefore it is of great importance to establish the correlation between Hsp27 and the Hippo pathway to further discover suitable targets in the treatment of metastatic malignancies. Methods: Hsp27 gain and loss of function experiments were done on 3 different cancer cell lines and the functional effects on every step of the pathway were monitored via Western blots and Immunofluorescence. Activity of YAP/TAZ after Hsp27 gain and loss of function was monitored by conducting qRT-PCR on TEAD target genes. Transcriptional activity of TEAD1 was also examined using a TEAD-dependent Luciferase reporter construct. Co-immunoprecipitation assay was conducted to analyze protein interactions in the absence/presence of Hsp27. Pathway activity in prostate cancer will be assessed by immunohistochemistry staining of core components of the pathway in patients’ tissue samples. Results: Our preliminary findings indicate that Hsp27 negatively affects the Hippo pathway. We found that targeting Hsp27 using siRNA in the PC3 (prostate cancer), A549 (lung cancer) and MDA-MB-453 (triple negative breast cancer) leads to increased cytoplasmic retention of p-YAP compared to control siRNA treated cells. Moreover, using immunofluorescence, we observed reduced nuclear translocation of the YAP/TAZ as well as sequestration of these components with cytoplasmic 14-3-3 proteins in siRNA treated PC3 cells. Furthermore inhibition of Hsp27 in prostate and lung tumour cells resulted in suppression of TEAD transcriptional activity analyzed by qRT-PCR and luciferase assay. Hsp27 over-expression experiments yielded opposite results compared to knockdown. Conclusion: Hsp27 overexpression contributes to inactivation of Hippo pathway. Targeting Hsp27 leads to inactivation of YAP and TAZ onco-proteins affecting cancer cell survival. Impact: Our data further supports the significance of targeting Hsp27 as a treatment option in cancers, especially metastatic malignancies like CRPC. Citation Format: Sepideh Vahid, Daksh Thaper, Amina Zoubeidi. Hsp27 negatively affects Hippo tumor suppressor pathway to regulate cell survival in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4956. doi:10.1158/1538-7445.AM2015-4956

Abstract 3162: NOD1 augments cancer cell metastatic potential through p38 MAP kinase activation

Abstract INTRODUCTION: Strong clinical evidence demonstrates a link between acute bacterial infection and metastasis. We and others have shown that activation of certain membrane pattern recognition receptors (PRRs), such as Toll-like receptors, on cancer cells can be implicated in this process. There is emerging data that a cytoplasmic PRR, the nucleotide oligomerization domain receptor 1 (NOD1), may also play an important and complementary role in the immune response to bacterial infection, however its role in cancer progression is entirely unknown. We sought to determine the influence of NOD1 activation on metastasis. METHODS: NOD1 expression in human and murine colon (HT29, MC38) and lung (A549, H59) cancer cells were confirmed using flow cytometry (FC) and immunoblotting (WB). A series of in vitro and in vivo functional assays, including adhesion, migration and hepatic intravital microscopy (IVM), was conducted to assess the effect of NOD1 activation and inhibition. C12-iE-DAP, a highly selective NOD1 ligand derived from gram-negative bacterial wall, was used to simulate NOD1-activation under infectious condition. ML130, a specific NOD1 inhibitor, was used to block C12-iE-DAP activation. Stable knockdown (KD) of NOD1 in HT29 and A549 cells were constructed with shRNA lentiviral transduction and the functional assays were thus repeated. The predominant signaling pathway, downstream cytokines and cell adhesion molecules of NOD1-activation were identified using WB in the presence of kinase inhibitors. RESULTS: WB and FC showed abundant NOD1 expression in all tested cell lines. Cancer cells stimulated with C12-iE-DAP doubled the in vitro adhesion to collagen I, IV and fibronectin, as well as in vitro migration, an effect completely attenuated with ML130 inhibition and NOD1 knockdown. Using a murine hepatic adhesion assay under IVM, we observed a doubling in the amount of in vivo capture of cancer cells within hepatic sinusoids of C57BL6 mice in the C12-iE-DAP group compared to control, an effect again abrogated by ML130 inhibition and NOD1 knockdown. Immunoblotting using HT29 cells under a panel of kinase inhibitors revealed that NOD1 activation is p38 dependent. Subsequently, the use of p38 inhibitor, BIRB0796, abolished C12-iE-DAP mediated adhesion to collagen I and fibronectin. Using human cytokine quantification array, we determined that NOD1 activation led to increase in IL-8, 15, MCP1 and RANTES, all of which have been shown to link to cancer progression or to chemoattract neutrophils, which we have recently shown to mediate metastasis. CONCLUSION: Our data demonstrate that NOD1 activation by the gram-negative bacterial wall component, C12-iE-DAP is important in enhancing the metastatic potential of cancer cells, and its mechanism is dependent on p38 activation and the upregulation of downstream cytokines. This is the first study to implicate NOD1 in metastasis, and thus identify this receptor as a putative therapeutic target. Citation Format: Henry Jiang, Sara Najmeh, Julie Berube, Arielle Leone, Paul Savage, Betty Giannias, France Bourdeau, Simon Rousseau, Morag Park, Lorenzo Edwin Ferri. NOD1 augments cancer cell metastatic potential through p38 MAP kinase activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2015-3162

Abstract P4-04-13: Cancer-derived miR-105 promotes tumor invasion and destroys the natural barriers against metastasis

Abstract Cancer-secreted miRNAs are emerging mediators of cancer–host crosstalk. In this study, we set out to identify cancer-derived miRNAs that participate in cancer metastasis by adapting the niche cells. We show that miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In epithelial and endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in non-metastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas intervention of miR-105 in highly metastatic tumors alleviates this effect. By testing clinical specimens from breast cancer patients, we further show that miR-105 can be detected in the circulation at the pre-metastatic stage, and its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer. Our results demonstrate for the first time the dual roles of miR-105 in inducing the migratory potential of cancer cells as well as destroying the epithelial and endothelial barriers in the cancer niche by targeting the cellular tight junctions. In breast cancer patients, increased levels of miR-105 in the circulation can be detected at the pre-metastatic stage and predict the occurrence of metastasis. Anti-miR-105 treatment suppresses metastasis and abolishes the systemic effect of tumor-derived miR-105 on niche adaptation. Therefore, these pre-clinical observations strongly suggest clinical applications of miR-105 as a predictive or early-diagnostic blood-borne marker as well as a therapeutic target for breast cancer metastasis. Citation Format: Shizhen Emily Wang, Weiying Zhou, Miranda Y Fong, Yongfen Min, Pengnian Charles Lin. Cancer-derived miR-105 promotes tumor invasion and destroys the natural barriers against metastasis [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 P4-04-13.

Abstract P6-10-04: Statins and breast cancer stage and mortality in the Women's Health Initiative cohort

Abstract Background: Statins are the most widely prescribed cholesterol-lowering drugs in the United States, with approximately 25% of US adults using statins by 2008. The anti-carcinogenic effect of statins may reduce the metastatic potential of cancer cells leading to ‘stage migration’ with statin users more likely diagnosed with early rather than late stage cancer. We evaluated the effects of statins on breast cancer stage migration and breast cancer-specific mortality in the Women’s Health Initiative (WHI) Clinical Trial and Observational Study. Methods: The study population included 128,675 postmenopausal women aged 50 to 79 years, with 7,883 newly-diagnosed pathologically-confirmed cases of in situ (19%), local stage (61%), regional stage (19%) and distant stage (1%) breast cancer and 401 deaths due to breast cancer after an average of 11.5 (SD=3.7) years of follow-up. To reduce the possibility of detection bias, we excluded women who did not report a mammogram within 5 years of study entry, and who had no health insurance or medical care provider (n=28,237). Stage was coded using criteria implemented in the Surveillance, Epidemiology and End Results Program and stratified into early stage (in situ and local) vs. late stage (regional and distant). Information on statin use prior to breast cancer diagnosis was collected at baseline and years one, three, six, and nine years post-baseline. Self- and interviewer-administered questionnaires were used to collect risk factor information. Cause of death was based on medical record review by physician adjudicators. Cox proportional hazards regression models were used to calculate hazard ratios (HR) with 95% confidence intervals (CI) to evaluate the relationship between statin use as a time-dependent exposure and diagnosis of late stage breast cancer and breast cancer-specific mortality adjusting for important confounders. For these models, participants who had early stage breast cancer or who died during follow-up were censored at time of diagnosis or death, respectively. We also evaluated the effect of statins stratified by estrogen receptor (ER) status. Statistical tests were two-sided. Results: Statins were used by 10,474 women (8%) at baseline. In the multivariable-adjusted time-dependent model, statin use was associated with a marginal reduction in risk of late stage breast cancer (HR 0.84, 95% CI: 0.70-1.02, p=0.082) and a reduction in risk of late stage estrogen receptor positive breast cancer (HR 0.79, 95% CI: 0.63-0.99, p=0.044). Statin use was also associated with a marginal reduction in breast cancer-specific mortality although it did not reach statistical significance (HR 0.59, 95% CI: 0.32-1.06, p=0.075). Conclusions: In the WHI, statin use was associated with a modest reduction in risk of late stage estrogen positive breast cancer and a non-significant decrease in breast cancer mortality. We are currently performing sensitivity analyses on these models. Studies using other large data sets with longer follow-up and information on cancer-directed therapy are needed to further evaluate this possible association. Citation Format: Monica P Arun, Pinkal Desai, Amy Lehman, Marilyn L Kwan, JoAnn E Manson, Sayeh Lavasani, Sylvia Wasswertheil-Smoller, Gloria E Sarto, Meryl S LeBoff, Jane Cauley, Michele L Cote, Jennifer Beebe-Dimmer, Allison Jay, Rowan T Chlebowski, Michael S Simon. Statins and breast cancer stage and mortality in the Women's Health Initiative cohort [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 P6-10-04.

Abstract A50: Crotamine derived fusion short-peptides and their effect on melanoma and breast cancer cells cultured in vitro

Abstract The search for molecules with therapeutic potential against cancer is a crucial issue to reduce the amount of death in human population around the world. Previously, we showed that crotamine, a natural polypeptide from the venom of the rattlesnake Crotalus durissus terrificus, has ability to induce cancer cells apoptosis in vitro, as well as, delay and inhibit melanomas formation in vivo in mice. In order to reduce crotamine length and toxicity, while maintaining its anti-cancer properties, two short peptides were designed and synthetized. Such short peptides represent an intermediate potential step toward to the development of drugs targeted cancer death. The aim of present study was to analyze pro-apoptotic activity of both of these peptides (1 and 2), composed by crotamine (crot.) and BAX (pro-apoptotic protein from BCL2 family) both derived sequences in melanoma and breast cancer cells in vitro. Melanoma and breast cancer cells (MEL-85, A2058, B16-F10 and SKBR3) were used. The MTT assay was performed after addition of both of these peptides used in different concentrations into cancer cells. The expression of proteins (PARP, Cleaved - Caspase 3, Caspase 3, Phospho - BAD) was evaluated by western blotting. Flow cytometry was performed to evaluate the cell cycle. The mitochondria potential was also verified before and after peptides addition. Morphologic changes, such as, detachment from substrate, cell contraction, loss of inter-cell elongations and several others effects were observed in cancer cells after 24 hours of incubation with peptides. At that time, the peptides exhibit cytotoxic activity as verified by MTT assay. Cell cycle analyses demonstrated that they decrease the timing of G2/M phases, while increasing a sub-G0/G1 phase. Unexpectedly, both peptides increase mitochondria potential of cancer cells, when compared with control cultures (without addition of peptides). Increase of expression pro-apoptotic proteins, such as, cleaved Caspase 3 and PARP was evidenced in cancer cells after addition of peptide -1, while Phospho-BAD was increased after addition of peptide - 2. Our data suggest that both peptides are efficient to trigger cell death in melanoma and breast cancer cells in vitro. Crot alone was not able to induce such changes in cancer cells. Citation Format: Nicole Caroline Mambelli, Paulo Luis De-Sá-Júnior, Jr., Adilson Kleber Ferreira, Ricardo Alexandre Azevedo, Diana Aparecida Câmara, Irina Kerkis, Sr.. Crotamine derived fusion short-peptides and their effect on melanoma and breast cancer cells cultured in vitro. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A50.

HU-331 is a catalytic inhibitor of topoisomerase IIα.

Topoisomerases are essential enzymes that are involved in DNA metabolism. Topoisomerase II generates transient DNA strand breaks that are stabilized by anticancer drugs, such as doxorubicin, causing an accumulation of DNA damage. However, doxorubicin causes cardiac toxicity and, like etoposide and other topoisomerase II-targeted agents, can induce DNA damage, resulting in secondary cancers. The cannabinoid quinone HU-331 has been identified as a potential anticancer drug that demonstrates more potency in cancer cells with less off-target toxicity than that of doxorubicin. Reports indicate that HU-331 does not promote cell death via apoptosis, cell cycle arrest, caspase activation, or DNA strand breaks. However, the precise mechanism of action is poorly understood. We employed biochemical assays to study the mechanism of action of HU-331 against purified topoisomerase IIα. These assays examined DNA binding, cleavage, ligation, relaxation, and ATPase activities of topoisomerase IIα. Our results demonstrate that HU-331 inhibits topoisomerase IIα-mediated DNA relaxation at micromolar levels. We find that HU-331 does not induce DNA strand breaks in vitro. When added prior to the DNA substrate, HU-331 blocks DNA cleavage and relaxation activities of topoisomerase IIα in a redox-sensitive manner. The action of HU-331 can be blocked, but not reversed, by the presence of dithiothreitol. Our results also show that HU-331 inhibits the ATPase activity of topoisomerase IIα using a noncompetitive mechanism. Preliminary binding studies also indicate that HU-331 decreases the ability of topoisomerase IIα to bind DNA. In summary, HU-331 inhibits relaxation activity without poisoning DNA cleavage. This action is sensitive to reducing agents and appears to involve noncompetitive inhibition of the ATPase activity and possibly inhibition of DNA binding. These studies provide a promising foundation for the exploration of HU-331 as a catalytic inhibitor of topoisomerase IIα.