Development Of Metastatic Tumors Research Articles

Bone marrow mesenchymal stem cells suppress metastatic tumor development in mouse by modulating immune system.

IntroductionBone marrow mesenchymal stem cells (BMSCs) have been studied extensively because of their potential use in clinical therapy, regenerative medicine, and tissue engineering. However, their application in tumor therapy remains yet in preclinical stage because of the distinct results from different researches and vagueness of their functional mechanism. In this study, the influence of BMSCs on tumor growth was observed and the potential mechanism was investigated.MethodTwo animal models, H22 ascitogenous hepatoma in BALb/c mouse and B16-F10 pulmonary metastatic melanoma in C57 mouse, were adopted in experience in vivo and treated with BMSCs by intravenous injection. The percentage of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSCs) and IFN-γ+ T cells were observed in peripheral blood (PB) and bone marrow (BM) by Flow Cytometry. BMSCs were co-cultured in vitro with tumor cells and MDSCs in a tumor conditioned medium separately in order to illustrate the mechanism.ResultsOur results demonstrated that BMSCs treatment caused a delayed tumor growth and a prolonged survival in both tumor models, the homing fraction of BMSCs in BM was 2% - 5% in 24–72 hours after transfusion and the percentage of Gr-1+CD11b+ MDSCs was downregulated in peripheral blood and BM. Meanwhile, IFN-γ+ T lymphocytes in PB increased. In vitro co-culture showed that BMSCs inhibited the induction and proliferation of MDSCs in tumor conditioned medium, whereas they didn’t affect the proliferation of B16-F10 and H22 cells by in vitro co-culture. Both in vivo and in vitro results showed that BMSCs have a systemic suppressive effect on MDSCs.ConclusionOur data suggest that BMSCs has suppressive effect on tumor and is feasible to be applied in cancer treatment. BMSCs inhibiting MDSCs induction and proliferation is likely one of the mechanism.

Heregulin-HER3-HER2 signaling promotes matrix metalloproteinase-dependent blood-brain-barrier transendothelial migration of human breast cancer cell lines.

HER2-positive breast tumors are associated with a high risk of brain relapse. HER3 is thought to be an indispensible signaling substrate for HER2 (encoded by ERBB2) and is induced in breast cancer-brain metastases, though the molecular mechanisms by which this oncogenic dimer promotes the development of brain metastases are still elusive. We studied the effects of the HER3-HER2 ligand, heregulin (neuregulin-1, broadly expressed in the brain), on luminal breast cancer cell lines in vitro. Treatment of SKBr3 (ERBB2-amplified), MDA-MB-361 (ERBB2-amplified, metastatic brain tumor-derived) and MCF7 (HER2-positive, not ERBB2-amplified) cells with exogenous heregulin increased proliferation and adhesive potential, concomitant with induction of cyclin D1 and ICAM-1, and suppression of p27. All three cell lines invaded through matrigel toward a heregulin chemotactic signal in transwell experiments, associated with activation of extracellular cathepsin B and matrix metalloproteinase-9 (MMP-9). Moreover, heregulin induced breast cancer cell transmigration across a tight barrier of primary human brain microvascular endothelia. This was dependent on the activity of HER2, HER3 and MMPs, and was completely abrogated by combination HER2-HER3 blockade using Herceptin® and the humanized HER3 monoclonal antibody, EV20. Collectively these data suggest mechanisms by which the HER3-HER2 dimer promotes development of metastatic tumors in the heregulin-rich brain microenvironment.

Role of TGFB1 polymorphism in the development of metastatic brain tumors in non-small cell lung cancer patients.

We conducted a case-control study to investigate the role of 3 single-nucleotide polymorphisms of the gene encoding transforming growth factor-b1 (TGFB1) in the development of metastatic brain tumors in non-small cell lung cancer patients. The polymorphisms in TGFB1 rs4803455, rs1800469, and rs1800470 were evaluated by polymerase chain reaction-restriction fragment length polymorphism. Odds ratios and their corresponding 95% confidence intervals were used to assess the influence of TGFB1 rs4803455, rs1800469, and rs1800470 on metastatic brain tumors. We found that cases were more likely to have a later disease stage when compared with control subjects, without brain metastasis. Individuals carrying the TGFB1 rs1800469 TT and CT+TT genotypes had an increased risk of developing brain metastasis compared with the rs1800469 CC genotype. Moreover, a significant interaction was observed between the rs1800469 polymorphism and disease stage. However, no significant association between polymorphisms rs4803455 and rs1800470 and the risk of developing brain metastasis were observed. We found that the TGFB1 rs1800469 polymorphism may be predictive biomarker for the risk of developing brain metastasis in non-small cell lung cancer patients.

Blockade of extracellular NM23 or its endothelial target slows breast cancer growth and metastasis.

Nucleoside Diphosphate Kinase (NDPK), described as NM23 a metastasis suppressor, is found in the culture medium of cancer cells lines suggesting that the kinase may have an extracellular role. We propose that extracellular NM23 released from breast cancers in vivo stimulates tumor cell migration, proliferation and endothelial cell angiogenesis in support of metastasis development. NM23 in the bloodstream of immunocompromised mice carrying human triple-negative breast cancers or in breast cancer patients was measured by ELISA. Primary and metastatic tumor development, the impact of blockade of NM23 and/or its stimulation of nucleotide receptors were measured using in vivo imaging. NM23 expression data in the Curtis breast dataset was examined to test our hypothesis that NM23 may play a mechanistic role in breast cancer development. SCID mice carrying metastatic MDA-MB-231Luc+ triple-negative human breast tumor cells elaborate NM23 into the circulation correlated with primary tumor growth. Treatment of mice with the NM23 inhibitor ellagic acid (EA) or the purinergic receptor antagonist MRS2179 slowed primary tumor growth. At 16 weeks following implantation, lung metastases were reduced in mice treated with EA, MRS2179 or the combination. Expression of NM23 in the Curtis breast dataset confirmed a likely role for NM23 in tumor metastasis. Extracellular NM23 may constitute both a biomarker and a therapeutic target in the management of breast cancer.

Abstract B33: Langerhans cells prevent melanocytic nevus development and transformation in mice.

Abstract The function of Langerhans cells (LCs) in the skin immune system is unclear, and a matter of intense debate. In different mouse models LCs have been shown to induce antitumor immunity or promote skin squamous cell carcinoma (SCC). Their role in the control or promotion of melanoma is not known. We assessed LC's role in the development of DMBA-TPA induced pigmented benign and transformed lesions in a novel mouse model. Transgenic HuLang-DTA+/- C57Bl/6 mice, which selectively ablate epidermal LCs, were crossed with C3H/HeN mice to generate F1 LC knockout (KO) and WT littermate controls. LC-KO mice developed 40% more and 2-fold larger melanocytic lesions than WT. Cell suspensions from LC-KO pigmented skin lesions contained increased CD4+ Foxp3+ regulatory T cells and CD11b-/loGr-1hi cells, consistent with an immunosuppressive microenvironment. CD11c+CD11b+Ly6C+ APCs or MHCII+ cell numbers were fewer in lesions from LC-KO as compared to WT mice. Nests of melanocytic cells were observed at a higher frequency in draining lymph nodes (dLNs) from LC-KO as compared to WT; DLN cells contained increased numbers of TRP2+ cells and routinely gave rise to proliferative melanocytic cell lines. Real-time PCR analysis of epidermal cDNA from TPA treated LC-KO, as compared to WT, demonstrated a profound loss of IL-10, IL-12p35 and IL-12p40, indicating LCs as the major source. Further, IL-23 steady state levels in the epidermis of LC-KO are increased greater than 3-fold over WT, indicating that LCs normally suppress keratinocyte production of IL-23. Thus, in contrast to SCC, LCs inhibit development of benign and metastatic pigmented tumors. This novel murine model will be useful in elucidating transformation mechanisms and identifying novel therapeutic targets that may prevent metastatic melanoma. Citation Format: Tahseen H. Nasti, Yuko Tsuruta, Kris McKay, Mohammad Athar, Craig Elmets, Laura Timares. Langerhans cells prevent melanocytic nevus development and transformation in mice.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B33. doi:10.1158/1538-7445.CHTME14-B33

An imagable and photothermal "Abraxane-like" nanodrug for combination cancer therapy to treat subcutaneous and metastatic breast tumors.

A new "Abraxane-like" nanotheranostic formulation self-assembled from three clinically approved agents, human serum albumin, paclitaxel, and indocyanine green, is developed. Utilizing such a newly formulated nanodrug, combined photothermal and chemotherapy is conducted under the guidance of imaging, not only effectively eliminating subcutaneous tumors, but also significantly inhibiting the development of metastatic tumors in this proof-of-concept study.

High Prevalence of Human Cytomegalovirus in Brain Metastases of Patients with Primary Breast and Colorectal Cancers

BACKGROUND: Brain metastases (BMs) develop by largely unknown mechanisms and cause major morbidity and mortality in patients with solid tumors. Human cytomegalovirus (HCMV) is frequently detected in tumor tissue from patients with different cancers. Here, we aimed to determine the prevalence and potential prognostic role of HCMV in BMs. METHODS: We obtained archived samples of BMs from 41 patients with breast cancer and 37 with colorectal cancer and paired primary tumor tissues from 13 and 12 patients in each respective group. In addition, primary breast cancer tissues from 15 patients were included. HCMV proteins were detected with an immunohistochemical technique and Western blot. HCMV nucleic acids were detected with TaqMan polymerase chain reaction (PCR) assay. RESULTS: HCMV proteins were abundantly expressed in 99% of BM specimens, and in 12 of 13 (92%) paired primary breast cancer specimens. All 12 paired colon cancer samples were positive for HCMV proteins. Protein staining was mainly confined to neoplastic cells. Western blot analysis detected an HCMV-IE reactive protein in 53% of breast cancer specimens, and PCR detected the presence of HCMV DNA and transcripts in 92% and 80% of samples, respectively. Patients with high-level expression of HCMV-IE proteins in their tumors had a shorter time to tumor progression and shorter overall survival. CONCLUSIONS: The prevalence of HCMV proteins and nucleic acids is very high in primary and metastatic tumors and may drive the development of metastatic brain tumors; therefore, this virus may represent a potential therapeutic target in metastatic cancer.

Raf kinase inhibitor protein (RKIP) deficiency decreases latency of tumorigenesis and increases metastasis in a murine genetic model of prostate cancer.

Raf kinase inhibitor protein (RKIP) has been shown to act as a metastasis suppressor gene in multiple models of cancer. Loss of RKIP expression promotes invasion and metastasis in cell transplantation animal models. However, it is unknown if RKIP expression can impact the progression of cancer in an autochthonous model of cancer. The goal of this study was to determine if loss of RKIP expression in a genetic mouse model of prostate cancer (PCa) impacts metastasis. Endogenous RKIP expression was measured in the primary tumors and metastases of transgenic adenocarcinoma of the mouse prostate (TRAMP(+) ) mice. RKIP knockout mice (RKIP(-/-) ) were crossbred with (TRAMP(+) ) mice to create RKIP(-/-) TRAMP(+) mice. Mice were euthanized at 10, 20, and 30 weeks for evaluation of primary and metastatic tumor development. To determine if loss of RKIP alone promotes metastasis, RKIP was knocked down in the low metastatic LNCaP prostate cancer cell line. Endogenous RKIP expression decreased in TRAMP(+) mice as tumors progressed. Primary tumors developed earlier in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice. At 30 weeks of age, distant metastases were identified only the RKIP(-/-) TRAMP(+) mice. While prostate epithelial cell proliferation rates were higher at 10 and 20 weeks in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice, by 30 weeks there was no difference. Apoptosis rates in both groups were similar at all timepoints. Decreased RKIP expression did not impact the metastatic rate of LNCaP in an orthotopic PCa model. These results demonstrate that loss of RKIP decreases latency of tumor development and promotes distant metastasis in the TRAMP mouse model in the context of a pro-metastatic background; but loss of RKIP alone is insufficient to promote metastasis. These findings suggest that in addition to its known metastasis suppressor activity, RKIP may promote tumor progression through enhancing tumor initiation. Prostate 75:292-302, 2015. © 2014 Wiley Periodicals, Inc.

Abstract 2475: Zpo2 promotes aggressive breast cancer development through downregulation of GATA3

Abstract Metastatic breast cancer is the major cause of poor prognosis and death in women. The transcription factor GATA3 is a key mediator of luminal cell fate determination and homeostasis in adult mammary gland. Loss of GATA3 has been associated with development of metastatic mammary tumors. Here we describe a novel role for transcriptional repressor, Zeppo2 (zinc finger elbow-related proline domain protein2; Zpo2) (ZNF503) in promoting metastatic breast cancer through modulation of GATA3. We demonstrate that Zpo2, through its interaction with repressor of GATA (ROG) is capable of modulating GATA3 transcription in mammary epithelial cells. Overexpression of Zpo2 results in upregulation of EMT associated genes, decreased cell-cell adhesion and increased cellular invasiveness in 3D cultures. Additionally, overexpression of Zpo2 in mammary tumor cells results in enhancement of tumor growth and increased metastasis in orthotopic transplant models. Furthermore, to investigate the importance of Zpo2 in human breast cancer metastasis, we utilized a series of new patient-derived xenograft (PDX) models (primary human xenograft models). The PDX models ranged from poorly metastatic to highly metastatic as was observed in the (affected) original patients. Interestingly, higher Zpo2 expression levels correlate with more aggressive xenograft tumor models. Collectively, our data suggests that Zpo2 plays a potentially significant role in initiating aggressive breast cancer by modulating GATA3 in mammary epithelial cells. Citation Format: Payam Shahi, Euan M. Slorach, Jonathan Chou, Devon Lawson, Ying Yu, Zena Werb. Zpo2 promotes aggressive breast cancer development through downregulation of GATA3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2475. doi:10.1158/1538-7445.AM2014-2475

TGFβ signaling promotes juvenile granulosa cell tumorigenesis by suppressing apoptosis.

Molecular changes that give rise to granulosa cell tumors of the ovary are not well understood. Previously, we showed that deletion in granulosa cells of the bone morphogenetic protein receptor-signaling transcription factors, Smad1 and Smad5, causes development of metastatic granulosa cell tumors that phenocopy the juvenile form of granulosa cell tumors (JGCTs) in humans. The TGFβ-SMAD2/3 pathway is active in JGCTs, but its role is unknown. We tested the in vivo contribution of TGFβ-SMAD signaling to JGCT development by genetically deleting the common Smad4 from Smad1/5 double knockout mice. Smad1/5/4 triple knockout mice were sterile and had significantly increased survival and delayed tumor development compared to those for the Smad1/5 double knockout mice. The few tumors that did develop were smaller, showed no evidence of metastasis, and had increased apoptosis. In the human JGCT cell line COV434, TGFβ1 increased viability by inhibiting apoptosis through a TGFβ type I receptor-dependent repression of caspase activity and inhibition of poly(ADP-ribose) polymerase cleavage. These data support a tumor-promoting function of TGFβ in JGCTs through its ability to repress apoptosis.

Cooperation of p53 mutations with other oncogenic alterations in cancer.

Following the initial findings suggesting a pro-oncogenic role for p53 point mutants, more than 30 years of research have unveiled the critical role exerted by these mutants in human cancer. A growing body of evidence, including mouse models and clinical data, has clearly demonstrated a connection between mutant p53 and the development of aggressive and metastatic tumors. Even if the molecular mechanisms underlying mutant p53 activities are still the object of intense scrutiny, it seems evident that full activation of its oncogenic role requires the functional interaction with other oncogenic alterations. p53 point mutants, with their pleiotropic effects, simultaneously activating several mechanisms of aggressiveness, are engaged in multiple cross-talk with a variety of other cancer-related processes, thus depicting a complex molecular landscape for the mutant p53 network. In this chapter revealing evidence illustrating different ways through which this cooperation may be achieved will be discussed. Considering the proposed role for mutant p53 as a driver of cancer aggressiveness, disarming mutant p53 function by uncoupling the cooperation with other oncogenic alterations, stands out as an exciting possibility for the development of novel anti-cancer therapies.

Disarming mutant p53 oncogenic function

In the last decade intensive research has confirmed the long standing hypothesis that some p53 point mutants acquire novel activities able to cooperate with oncogenic mechanisms. Particular attention has attracted the ability of several such mutants to actively promote the development of aggressive and metastatic tumors in vivo. This knowledge opens a new dimension on rational therapy design, suggesting novel strategies based on pharmacological manipulation of those neomorphic activities. P53 point mutants have several characteristics that make them attractive targets for anti-cancer therapies. Remarkably, mutant p53 has been found predominantly in tumor cells and may act pleiotropically by interfering with a variety of cellular processes. Therefore, drugs targeting mutant p53 may selectively affect tumor cells, inactivating simultaneously several mechanisms of tumor promotion. Moreover, the high frequency of missense mutations on the p53 gene suggests that interfering with mutant p53 function may provide a valuable approach for the development of efficient therapies able to target a wide range of tumor types.

Brain Neoplasms and Coagulation

Brain vasculature is uniquely programmed to protect central nervous system tissues and respond to their metabolic demands. These functions are subverted during the development of primary and metastatic brain tumors, resulting in vascular perturbations that are thought to contribute to progression and comorbidities of the underlying disease, including thrombosis and hemorrhage. Chronic activation of the coagulation system is particularly obvious in glioblastoma multiforme (GBM), where intratumoral vasoocclusive thrombosis may contribute to hypoxia, pseudopalisading necrosis, and angiogenesis. GBM is also associated with spontaneous or iatrogenic bleeding, and the emission of circulating procoagulants implicated in the unusually high risk of peripheral venous thromboembolism. Tissue factor (TF) expression is elevated in several types of brain tumors, including adult and pediatric GBM, as is the production of TF-containing microparticles (TF-MPs). Both TF expression and its vesicular emission are regulated by tumor microenvironment (e.g., hypoxia), in concert with activated oncogenic and growth factor pathways (RAS, EGFR, MET), as well as the loss of tumor suppressor gene activity (PTEN). Discovery of distinct oncogenic networks led to recognition of unique molecular subtypes within brain tumors, of which GBM (proneural, neural, classical, and mesenchymal), and medulloblastoma (SHH, WNT, group 3, and group 4) exhibit subtype-specific composition of the tumor coagulome. It remains to be established whether mechanisms of thrombosis and biological effects of coagulation in brain tumors are also subtype specific. In this regard, TF pathway represents a paradigm, and its impact on tumor dormancy, inflammation, angiogenesis, formation of cancer stem cell niches, and dissemination is a subject of considerable interest. However, establishing the extent to which TF and TF-MPs contribute to pathogenesis and thromboembolic disease in the context of primary and secondary brain tumors may require molecular stratification of patient populations. We suggest that a better understanding of these molecular linkages may pave the way to a more effective (targeted) therapy, prophylaxis, adjunctive use of anticoagulants, and other agents able to modulate interactions between brain tumors and the coagulation system.

Characteristics and survival of patients with advanced cancer and TP53 mutations in phase I clinical trials.

2545 Background: The classical hot spot mutations within the central sequence-specific DNA-binding region of p53 protein (R175H in exon 5 and G245S, R248Q, R249S, R273H and R282W in exon 8) disrupt its ability to bind to DNA. These inactivating point mutations of p53 may result in loss of its function and/or acquisition of a gain of function, which has been associated with the development of invasive and metastatic tumors in mouse models. We assessed the frequency and survival of p53 mutations in patients with advanced cancer. Methods: P53 mutation in patients’ tumor was identified in a CLIA-certified laboratory using PCR, sequenom analysis or next generation sequencing. Results: P53 mutations were identified in 212 patients. Median survival by tumor type and distribution of p53 mutations by exon are shown in the Table. The median survival (from time of referral to Phase I clinic) by mutations in “hot spot” amino acids was as follows: R175H, 9.1 months (n=18); R248Q, not reached/5 deaths (n=22); R273H, 12.6 months (n=11); other mutations, 11.9 months (n=160). The median survival of patients with a tumor mutation in exons 5 or 8 appeared to be shorter than that of patients with mutations in other exons (9.4 vs. 14.6 months) although the difference was not statistically significant (p=.12) owing to the small number of patients. Conclusions: Our results demonstrate that the distribution and clinical significance of various p53 mutations differs by tumor type and suggest that mutations in R248Q may be associated with longer survival. [Table: see text]

Supplementation with Selenium-Enriched Yeast Attenuates Brain Metastatic Growth

Metastases are the leading cause of cancer mortality and their development may be affected by diet. The aim of this study was to compare the effects of dietary supplementation with different selenium (Se) compounds on the dynamics of brain metastasis development in a novel mouse model. Mice were fed experimental diets enriched (1 mg/kg) with sodium selenite (Se-S), seleno-1-methionine (Se-Meth), a yeast-derived organic form of selenium (Se-Yeast), or a control diet (Se < 0.05 mg/kg) for 20 wk. At the end of the feeding period, animals were injected with luciferase-tagged K1735 (K1735-Luc) melanoma cells into the brain vasculature. The development of brain metastatic tumors was monitored for 2 wk following injection. Mice bearing brain metastatic tumors and fed Se-Yeast- or Se-S-enriched diets displayed a higher survival rate compared with other experimental and control groups. Importantly, Se-Yeast supplementation decreased the growth of brain metastatic tumors as determined by the measurement of the intensity of the bioluminescent signal emitted by K1735-Luc cells upon reaction with luciferin. Different chemical forms of Se have distinct effects on the development of brain metastases. Organic Se in the form of Se-Yeast may be a valuable agent in suppression of brain metastatic disease.

Abstract LB-285: Pro-tumor N2 neutrophils contribute to the pre-metastatic niche.

Abstract Metastasis is the primary cause of cancer-related mortalities and the tissue microenvironment that supports tumor cell attachment and growth at metastatic sites has become an active area of investigation. Here we present evidence that pro-tumor N2 neutrophils are one of the bone marrow-derived cell types that establish this pre-metastatic niche. In the murine Lewis Lung adenocarcinoma (LLC) in vivo metastasis model, tumor cells metastasize to the lungs with increasing primary tumor growth over time. In these studies, immunocompetent mice injected subcutaneously with syngeneic LLC cells are sacrificed sequentially 10-32 days later, before and after established lung metastases. Neutrophil chemoattractants KC/CXCL1 and MIP-2/CXCL2 are significantly elevated in pre-metastatic tissue compared to normal lungs of healthy contemporary controls. MCP-1/CCL2, a marker of pro-tumor N2 neutrophils, increases steadily over time in pre-metastatic lung tissue of study mice and is significantly elevated in pre-metastatic tissue and in metastases compared to normal lung tissue. Isolated lung-infiltrating neutrophils also express N2 markers. In contrast, TNF-alpha, a marker for anti-tumor N1 cells, remains low or undetectable in lung tissue throughout metastatic tumor development. Neither active nor total TGF-beta, the putative control switch for a pro-tumor phenotype, is elevated in pre-metastatic tissue. Notably, levels of all cytokines are low or undetectable in normal lungs from naïve mice and in other organs of tumor-bearing mice (non-metastatic sites). Ly6G+ neutrophils were detected at a higher density in the lungs of pre-metastatic and metastatic mice compared to normal lungs, although the density of myeloperoxidase (MPO)-stained cells was similar in all groups. These data are consistent with recent reports that MPO activity is low or absent from tumor-associated neutrophils, although high in MDSC and naïve neutrophils. Neutrophil distribution in metastatic lung tissue is most concentrated around the tumor foci and tumor vasculature. Finally, studies of lung tissue from mice bearing LLC variants with different metastatic capacities are also consistent with our premise that neutrophils help establish the pre-metastatic niche. Understanding the factors that regulate the development and growth of malignant cells at metastatic sites is critical for improved clinical strategies in the prevention, diagnosis and treatment of metastases. Citation Format: Jered Cope Meyers, H. Keith Pittman, Hunter Story, Dare M. Imes, George A. Howard, Kathryn M. Verbanac. Pro-tumor N2 neutrophils contribute to the pre-metastatic niche. [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 LB-285. doi:10.1158/1538-7445.AM2013-LB-285

Abstract 4902: A putative stem cell marker, doublecortin-like kinase (DCLK1), is involved in the development of hepatocellular carcinoma.

Abstract Introduction: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. We recently demonstrated that expression of doublecortin-like kinase DCLK1, also called DCAMKL1, tend to increase as the liver condition advances from normal to cirrhosis and HCC. A significant increase in the number of DCLK1+ cells is observed in patients who develop low-grade cirrhosis to dysplastic regenerative nodules. DCLK1 is a putative intestinal stem cell marker that is overexpressed in cells with tumor-initiating properties and regulates tumor growth and tumor-suppressor miRNAs. Therefore, we investigated the role of DCLK1 in HCC. Methods: Normal human hepatocytes (NHHs) were cultured in Matrigel containing Hepato-STIM media supplemented with epidermal growth factor. The NHH-derived 3D spheroids were analyzed for expression of phenotype markers and their ability to form tumor in immunodeficient mouse model. Cultured hepatoma cells (Huh7.5) were transplanted into the flanks of athymic nude to generate HCC-like tumor. The tumors were treated with PLGA nanoparticles (NPs) containing siRNA against DCLK1 (siDCLK1-NP) or scrambled siRNA (siSCR-NP) for two weeks. The tumor growth was monitored and cancer-related molecular markers in these tumors were analyzed after euthanizing the animals. Results: The NHH-derived 3D spheroids exhibited eccentric perinuclear staining of DCLK1 in most cells at early stages (2-3 weeks). However, asymmetric ductules associated with the spheroids lacked DCLK1 expression. Upon further growth, heterogeneous hepatic cell lineages appeared in the spheroids. The presence of hepatoblasts was characterized by co-staining of α-fetoprotein (AFP) with CK19. Other cell populations showed DCLK1 expression or the presence of cytoplasmic/membranous CK19. A number of cells in the spheroids were negative for CK19, AFP or DCLK1 whereas a few cells were positive for human albumin. Transplantation of these spheroids in the dorsal flanks of immunodeficient mice exhibited metastatic tumor development at multiple sites. The small cuboidal morphology, Alb- phenotypes, and presence of sporadic human Alb+ cells in the tumors suggest that most cells possessed undifferentiated characteristics. These tumors were highly enriched in DCLK1+ cells located near vascular areas. The treatment of Hu7.5-derived tumors with siDCLK1-NP resulted in a significant reduction in DCLK1 expression and concomitant decrease (∼60%) in tumor volume, whereas tumor growth was unaffected in the control animals treated with siSCR-NP. Conclusions: These observations indicate that hepatic DCLK1+ present in the early stages of spheroids may possess stem-like properties. DCLK1 appears to regulate tumorigenic behavior of hepatocytes and its down regulation leads to inhibition of cancer growth. Thus, DCLK1 is likely to be a novel target for HCC treatment. Citation Format: Naushad Ali. A putative stem cell marker, doublecortin-like kinase (DCLK1), is involved in the development of hepatocellular carcinoma. [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 4902. doi:10.1158/1538-7445.AM2013-4902

Intraosseous inoculation of tumor cells into bone marrow promotes distant metastatic tumor development: A novel tool for mechanistic and therapeutic studies

Bone marrow-derived cells have a potent impact on the formation and progression of tumor metastasis. This study demonstrates that bone marrow directly promotes metastasis to distant sites from tumor cells residing in the bone marrow in multiple types of tumors and multiple mouse strains. The bone marrow environment requires less tumor cells for inducing distant metastasis and overcomes the inhibition of metastasis resulting from engineering the tumor cells with reporter genes. This discovery provides an effective approach to generate spontaneous-like metastatic tumor models which will satisfy the urgent need for studying metastasis biology and discovering novel therapeutics.

Analysis of tumor‐induced lymphangiogenesis and lymphatic vessel invasion of pancreatic carcinoma in the peripheral nerve plexus

Cancer cells can metastasize throughout the body by various mechanisms, including the lymphatic system, resulting in tumor-induced lymphangiogenesis that can profoundly affect patient survival. The aim of the present study was to examine the role of lymphangiogenesis in the metastasis of pancreatic cancer to the peripheral nerve plexus. Immunohistochemistry was performed to analyze specimens obtained from 70 ductal adenocarcinoma patients. The markers used included lymphangiogenic factor vascular endothelial growth factor (VEGF)-C, the lymphatic-specific marker D2-40, and cytokeratin 19, an independent prognostic factor for pancreatic tumors. The relationship between survival rate and invasion of both the lymphatic vessels and peripancreatic nerve plexus (PNP) was evaluated, with clearly elevated lymphatic vessel density (LVD) in tissues adjacent to the cancer tissues. In fact, LVD levels were higher in adjacent tissues than in localized cancer tissues, and lymphatic vessel invasion into tissues adjacent to the tumor was significantly correlated with both PNP invasion (P = 0.005) and lymph node metastasis (P = 0.010). Correspondingly, LVD in tissues adjacent to the tumor was correlated with both invasion of lymphatic vessels surrounding the tumor (P = 0.024) and VEGF-C expression (P = 0.031); in addition, VEGF-C expression was correlated with invasion of lymphatic vessels around the tumor (P = 0.004). Survival rates were significantly lower in patients in whom there was peritumor lymphatic vessel invasion (P < 0.001), extrapancreatic nerve plexus invasion (P = 0.001), and/or lymph node metastasis (P < 0.001). Based on these results, lymphatic invasion associated with adjacent tumor growth likely contributes to the development of metastatic tumors that invade the PNP.

Interaction of FGF1 with a novel anti-angiogenic drug SSR128129E

Angiogenesis is the cellular process of formation of new blood vessels from preexisting vessels. This process is crucial in the development and progression of metastatic tumors. During chemotherapy treatments, cancer cells release large quantities of proteins (e.g., FGF1, FGF2, VEGF etc.) that induce angiogenesis and reduce the efficiency of chemotherapy. Thus, small molecular chemical antagonists of these proteins may represent an attractive new class of angiogenesis and lymphangiogenesis inhibitors in cancer therapies. In this manuscript, we report the study of the molecular interactions between acidic fibroblast growth factor (FGF1) and an antiangiogenic drug, SSR128129E [2-amino-5-(1-(1-methoxy-2-methylindolizin-3-yl)ethanone) benzoic acid]. 1H-15N HSQC titrations revealed that two SSR128129E molecules interact with FGF1. We solved the solution structure of the FGF1:SSR128129E (1:2) complex. No significant conformational changes were observed in FGF1 upon binding to SSR128129E by both NMR and circular dichroism. We also studied the interaction between SSR128129E and the D2 domain of FGFR by 1H-15N HSQC titration. SSR128129E binds to FGF1 as well as to the interface region of the D2 domain of its receptor, FGFR, preventing the formation of the FGF1-FGFR signaling complex.