Modulate Tumor Growth Research Articles

Abstract 253: IMP1 mediates tumor size and dissemination into the blood in colorectal cancer cell xenografts.

Abstract Introduction Colorectal cancer (CRC) remains amongst the top causes of cancer-related deaths in the United Sates, with nearly 60,000 deaths per year. Despite significant emphasis placed on improving early detection, only 40% of new CRC cases are diagnosed with localized-stage disease, which nonetheless is associated with a 5-year survival rate of 90%. However, the 5-year survival rate for regional and distal metastases is 68% and 11%, respectively, underscoring the importance of elucidating the underlying mechanisms for these invasive processes. Several recent studies support the emerging paradigm that the same factors that promote “stemness” in embryonic or adult stem cells may also play a critical role in tumorigenesis. We hypothesize that Insulin-like growth factor II mRNA binding protein 1 (IMP1) may be such a factor in the intestine. Imp1−/− mice exhibit dwarfism and increased perinatal lethality due to impaired gut formation, suggesting a potential role in stem cell maintenance in the intestine. Our previous data demonstrate that IMP1 loss decreases proliferation and anchorage-independent growth, and increases apoptosis in CRC cell lines. We hypothesize that IMP1 modulates tumor growth in vivo, and its overexpression may enhance disease progression. Methods In the current study, we generated stable CRC cell lines with IMP-1 overexpression and performed xenograft experiments. Furthermore, because IMP-1 overexpression in human CRC tumors correlates with increased metastasis, we used FACS to assess tumor dissemination into the blood. Finally, in order to determine mechanisms attributable to IMP1 and enhanced metastasis, we examined whether IMP1 modulates a tumor-initiating cell phenotype. Results Both control and IMP1 overexpressing tumors exhibited features of poorly differentiated carcinoma, and IMP1 overexpressing cells grew larger tumors. Mice harboring IMP1 overexpressing tumors had more tumor cells circulating in the blood at sacrifice. We did not observe gross metastases within the 8-week time course of xenograft tumor growth, however we cannot discount the possibility of establishment of micro-metastases. IMP1 overexpression led to a dramatic increase in CD24+CD44+ cells in vitro, suggesting that IMP1 may promote a tumor-initiating cell phenotype. Conclusions Our data suggest that IMP1 may play an important role in regulating primary tumor size as well as the ability of tumor cells to disseminate into the blood, possibly through the pro-survival advantage imparted by enhancing a tumor-initiating cell phenotype. Future studies will be required to assess whether the IMP1-mediated increase in tumor-initiating cells alone promotes metastasis, or whether additional factors are needed to establish tumor cells in distal sites. Funding: NIH F32 DK093207-01, NIH R01 DK056645-12, NIH U01 DK007066 Citation Format: Kathryn E. Hamilton, Prateek S. Katti, Christopher M. Hahn, Sonya R. Davey, Emma T. Lundsmith, Andrew D. Rhim, Anil K. Rustgi. IMP1 mediates tumor size and dissemination into the blood in colorectal cancer cell xenografts. [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 253. doi:10.1158/1538-7445.AM2013-253

Role of androgen and vitamin D receptors in endothelial cells from benign and malignant human prostate

Forty years ago, Judah Folkman (Folkman. N Engl J Med 285: 1182-1186, 1971) proposed that tumor growth might be controlled by limiting formation of new blood vessels (angiogenesis) needed to supply a growing tumor with oxygen and nutrients. To this end, numerous "antiangiogenic" agents have been developed and tested for therapeutic efficacy in cancer patients, including prostate cancer (CaP) patients, with limited success. Despite the lack of clinical efficacy of lead anti-angiogenic therapeutics in CaP patients, recent published evidence continues to support the idea that prostate tumor vasculature provides a reasonable target for development of new therapeutics. Particularly relevant to antiangiogenic therapies targeted to the prostate is the observation that specific hormones can affect the survival and vascular function of prostate endothelial cells within normal and malignant prostate tissues. Here, we review the evidence demonstrating that both androgen(s) and vitamin D significantly impact the growth and survival of endothelial cells residing within prostate cancer and that systemic changes in circulating androgen or vitamin D drastically affect blood flow and vascularity of prostate tissue. Furthermore, recent evidence will be discussed about the expression of the receptors for both androgen and vitamin D in prostate endothelial cells that argues for direct effects of these hormone-activated receptors on the biology of endothelial cells. Based on this literature, we propose that prostate tumor vasculature represents an unexplored target for modulation of tumor growth. A better understanding of androgen and vitamin D effects on prostate endothelial cells will support development of more effective angiogenesis-targeting therapeutics for CaP patients.

OP-11 Serum C-reactive protein (CRP) as biomarker for overall survival in patients with sorafenib therapy with hepatocellular carcinoma

treat several diseases. In particular, a major biological activity of G. frondosa is its antitumor activity, which has been a target for scientific and clinical research. G. frondosa is a mushroom indigenous to many Asian countries; it is an edible fungus with a large fruiting body characterized by overlapping caps. Liver cancer is a leading cause of cancer deaths, for which there is currently no successful chemotherapy. The aim of this study is to investigate the chemopreventive effects of G. frondosa in a liver cancer cell-line, Hep3B. Recent studies have revealed that G. frondosa inhibits tumor growth by inducing apoptosis. Methods: To investigate the inhibition of cell growth and ability by GFW treatment, we used a MTT assay, colony formation assay, soft agar assay and wound healing assay. Further, we also made use of phase contrast microscopy, flow cytometry and Western blot to detect autophagy and apoptosis. We next examined the ability of GFW to modulate tumor growth in xenograft mouse model. Results: This study examined the effect of G. frondosa on viability and apoptosis of human liver cancer cells Hep3B. We found that treatment with water extract of G. frondosa (GFW) (15.6 or 7.8 μg/mL) significantly reduced Hep3B cancer cells growth by 95% and 90% respectively, and induced cytoplasmic vacuolations. The autophagy-associated proteins LC3A, LC3B, ATG3, ATG5, ATG7, and Beclin-1 were also increased in Hep3B cancer cells treated with GFW. GFW inhibited the phosphorylation of PI3K (Tyr458/Tyr199) an important maker in autophagy signaling pathway, in addition, GFW increased the phosphorylation of JNK (Thr183/Tyr185), and down-regulation of Bcl-2. Moreover, prolonged exposure of Hep3B cells to GFW eventually caused apoptosis which was associated with suppression of phosphorylated Akt (Ser473) and ERK (Thr202/Tyr204), and activation of caspase-3 and -9, and concomitant degradation of PARP. We also demonstrated a GFW-induced therapeutic effect on Hep3B tumor development in nude mice. The feeding of GFW (50 mg kg–1 day–1) significantly inhibited tumor growth by 77.4%, as compared with the control group, in nude mice inoculated with Hep3B cancer cells. Conclusion: In conclusion, our results demonstrated that the PI3K signaling pathway promotes autophagy in GFW treated Hep3B cells. On the other hand, we also found that inhibition of ERK and of the PKC-delta signaling pathway promotes apoptosis in GFW-treated Hep3B cells. These findings implied that GFW may be used as a novel therapeutic reagent for the treatment of hepatocellular carcinoma.

Prion protein signaling as therapeutic target in human tumors.

Prions are proteinaceus infectious agents associated to invariable fatal neurodegenerative disorders named transmissible spongiform encephalopathies or prion diseases. The mechanism associated with disease propagation lays on post-translation modifications of the cellular prion protein (PrPC), which acquires altered conformation, form aggregates and is resistant to proteolysis. PrPC is a cell surface glycosyphosphatidylinositol (GPI)-anchored protein of 208-209 aminoacids which is codified by a single exon and whose expression is developmentally regulated. In adult animals PrPC is highly expressed in the central nervous system but peripheral nervous system and other organs or tissues also express this protein. Initial experiments conducted with PrPC-null mice established that this protein is absolutely necessary to propagate prion infection. Remarkably, these animals had no gross anatomical abnormalities or behavior alterations although higher neuronal sensitivity to stress conditions such us seizures and hypoxic-ischemic injury are present. These data pointed that PrPC would be unnecessary for embryonic development or some compensatory mechanism might be present in these animals. In fact, the later hypothesis was supported by findings that demonstrated that deletion of specific PrPC domains cause severe neurodegeneration in mice. The use of cellular models led to the characterization of PrPC ligands and related signaling pathways allowing the definition of PrPC cellular functions. We identified three major ligands for PrPC, two proteins of the extracellular matrix, laminin and vitronectin, and a secreted co-chaperone the stress inducible protein 1 (STI1) or Hop (its human homologue). Comparative experiments using neurons derived from wild type and PrPC-null mice demonstrated that PrPC binding to vitronectin promoted axonal growth in dorsal root ganglion neurons (DRG). In addition, PrPC interaction with laminin activated metabotropic glutamate receptors, Ca2+ mobilization and PKC activation promoting neuronal plasticity and memory formation in rats. STI1 was the most explored PrPC ligand. The PrPC-STI1 engagement induces PKA an ERK1/2 activation, which increase neuronal survival and differentiation. The signaling pathways modulated by PrPC-STI1 also included Ca2+ influx by activation of α-7 nicotinic acetylcholine receptor, induction of PI3K and mTOR. These last two pathways were associated to increased protein synthesis. An increment on the self-renewed of neuronal progenitors is also modulated by PrPC-STI1 engagement. The co-chaperone STI1 is known to interact with PrPC at the neuronal membrane and recently we demonstrated that this protein is abundantly secreted by astrocytes. Secreted STI1 plays an autocrine activity upon astrocytes modulating their proliferation and differentiation. The mechanism associated with STI1 secretion is under evaluation but preliminary results indicated that this protein is secreted by microvesicles derived from multivesicular bodies. The involvement of PrPC and STI1/Hop in cellular survival and differentiation raised questions regarding their involvement in tumoral processes. In fact, the literature has been pointed that PrPC expression contributes to cancer progression and resistance to various cancer therapies. STI1/Hop expression was also associated with the proliferation of tumor cells. Our data pointed that STI1 secreted by glioblastoma cells has an autocrine function mediating proliferation of these cells by binding to PrPC. We are presently evaluating how to interfere with the PrPC-STI1 binding in order to modulate tumor growth. Together our results point that PrPC-STI1 engagement is an interesting therapeutic target for both neurodegeneration and cancer and investments should be done to address these issues.

BMP5 treatment of adrenocortical carcinoma delays tumor growth via activation of endogenous anti-tumoral macrophages

Bone morphogenetic proteins (BMPs) are known to affect a variety of biological processes and also have been reported to modulate tumor growth. Former investigations revealed loss or downregulation of BMP5 in adrenocortical cancer (ACC) samples compared to normal adrenal glands and a deficit of downstream signalling in the human ACC cell line NCI h295R. Nude mice were subcutaneously xenografted with NCI h295R cells to establish solid tumors and treated with intra-tumoral administration of human recombinant (hr) BMP5 protein. Upon BMP5 delivery we observed a significant reduction in tumor size in comparison to PBS treated animals and a reduced proliferation rate (Ki-67 index) was evident. The canonical SMAD1/5/8 signalling pathway was not relevant in total tumor tissue with no detectable changes in human ID1, ID3, SMAD 6/7 and SMURF gene expression and SMAD1/5/8 phosphorylation. As BMP6 is reported to activate macrophages towards a pro-inflammatory anti-tumoral phenotype we were interested in a possible activating effect of BMP5. Expression of murine IL-12p40 was upregulated and a slight increase in the anti-angiogenic chemokines Mig (CXCL-9), IP-10 (CXCL-10) and murine macrophage marker F4/80 was detectable in BMP5 treated total tumor tissue. We also detected an increase in human CCL-2 (MCP1) expression, a monocyte attractant protein. Immunohistochemistry for galectin-3 showed influx of inflammatory macrophage-like cells into tumor tissue following BMP5 delivery. Murine macrophage cells (RAW 264.7) were treated with hrBMP5 and an increase in murine TNF-α expression and in the expression of the differentiation-related macrophage transcription factors PU.1 and IRF-5 was detected as well as upregulation of murine ID1 and ID3. We assume that the BMP5 mediated reduction in tumor growth might be achieved not via intracellular BMP signalling but rather by activation of anti-tumoral macrophages exerting anti-angiogenic, cytostatic or cytotoxic function on ACC tumors.

BM mesenchymal stromal cell–derived exosomes facilitate multiple myeloma progression

BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC-derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC-derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression.

Predictive pharmacokinetic–pharmacodynamic modeling of tumor growth after administration of an anti-angiogenic agent, bevacizumab, as single-agent and combination therapy in tumor xenografts

Pharmacokinetic-pharmacodynamic (PK-PD) models able to predict the action of anticancer compounds in tumor xenografts have an important impact on drug development. In case of anti-angiogenic compounds, many of the available models show difficulties in their applications, as they are based on a cell kill hypothesis, while these drugs act on the tumor vascularization, without a direct tumor cell kill effect. For this reason, a PK-PD model able to describe the tumor growth modulation following treatment with a cytostatic therapy, as opposed to a cytotoxic treatment, is proposed here. Untreated tumor growth was described using an exponential growth phase followed by a linear one. The effect of anti-angiogenic compounds was implemented using an inhibitory effect on the growth function. The model was tested on a number of experiments in tumor-bearing mice given the anti-angiogenic drug bevacizumab either alone or in combination with another investigational compound. Nonlinear regression techniques were used for estimating the model parameters. The model successfully captured the tumor growth data following different bevacizumab dosing regimens, allowing to estimate experiment-independent parameters. A combination model was also developed under a 'no-interaction' assumption to assess the effect of the combination of bevacizumab with a target-oriented agent. The observation of a significant difference between model-predicted and observed tumor growth curves was suggestive of the presence of a pharmacological interaction that was further accommodated into the model. This approach can be used for optimizing the design of preclinical experiments. With all the inherent limitations, the estimated experiment-independent model parameters can be used to provide useful indications for the single-agent and combination regimens to be explored in the subsequent development phases.

GRANULOCYTE INFILTRATION AND EXPRESSION OF THE PRO-ANGIOGENIC BV8 PROTEIN IN EXPERIMENTAL EL4 AND LEWIS LUNG CARCINOMA TUMORS.

Although Vascular Endothelial Growth Factor (VEGF)-targeted therapies have shown efficacy in the treatment of certain advanced cancers, benefits to patients have been modest, which is attributed to tumor resistance to VEGF neutralization. Recent efforts to identify new targets to inhibit tumor angiogenesis have identified Bv8 (prokineticin 2), a myeloid cell-derived protein that promotes endothelial cell growth and tumor angiogenesis, but many mechanistic aspects of the pro-tumorigenic function of Bv8 are unclear. Here we demonstrate that CD11b+, Ly6C+, Ly6G+ granulocytes are the predominant cell source of Bv8 expression in bone marrow, spleen and in tumor tissues. Using granulocyte-deficient Growth factor independence-1 (Gfi1)-null mutant mice and normal littermates, we found that EL4 lymphoma tumors grow significantly larger in the granulocyte and Bv8-deficient mutant mice in comparison to the normal mice that display abundant tumor-associated granulocytes and Bv8 expression. Conversely, Lewis lung carcinoma (LLC-1) tumors grew to a significantly greater size in the normal mice in comparison to the Gfi1-null mice, but normal granulocyte tumor infiltration was modest. Quantitative analysis of tissue vascularization showed that EL4 and LLC-1 tumors from normal and Gfi1-mutant mice are similarly vascularized. These results confirm the critical contribution of the tumor microenvironment in determining the rate of tumor progression independently of tumor angiogenesis, and reveal some of the complexities of granulocyte and Bv8 functions in modulating tumor growth.

Mechanical Pressure Arrests the Growth of Tumor Spheroids

Often tumors have to push their surroundings in order to grow. Thus, during their development, tumors must be able to both exert and sustain mechanical stresses. We study quantitatively the effect of an applied mechanical stress on the long-term growth of a spherical cell aggregate. Our results indicate the possibility to modulate tumor growth depending on the applied pressure. Moreover, we observe that a stress between 500 and 5000 Pa drastically reduces growth by inhibition of cell proliferation mainly in the core of the spheroid, while it sligthly affect the division at the perifery.FIGURE: Three cryosections of a spheroid grown for 2 days without stress, with a stress of 1 kPa or with a stress of 5 kPa. Dividing cells are stained incyan (Antibody against Ki67).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Cav1 Suppresses Tumor Growth and Metastasis in a Murine Model of Cutaneous SCC through Modulation of MAPK/AP-1 Activation

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Receptor-binding domain of ephrin-A1: Production in bacterial expression system and activity

Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

A human monoclonal antibody 264RAD targeting αvβ6 integrin reduces tumour growth and metastasis, and modulates key biomarkers in vivo

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis.

Immunoediting: evidence of the multifaceted role of the immune system in self-metastatic tumor growth

BackgroundThe role of the immune system in tumor progression has been a subject for discussion for many decades. Numerous studies suggest that a low immune response might be beneficial, if not necessary, for tumor growth, and only a strong immune response can counter tumor growth and thus inhibit progression.MethodsWe implement a cellular automaton model previously described that captures the dynamical interactions between the cancer stem and non-stem cell populations of a tumor through a process of self-metastasis. By overlaying on this model the diffusion of immune reactants into the tumor from a peripheral source to target cells, we simulate the process of immune-system-induced cell kill on tumor progression.ResultsA low cytotoxic immune reaction continuously kills cancer cells and, although at a low rate, thereby causes the liberation of space-constrained cancer stem cells to drive self-metastatic progression and continued tumor growth. With increasing immune system strength, however, tumor growth peaks, and then eventually falls below the intrinsic tumor sizes observed without an immune response. With this increasing immune response the number and proportion of cancer stem cells monotonically increases, implicating an additional unexpected consequence, that of cancer stem cell selection, to the immune response.ConclusionsCancer stem cells and immune cytotoxicity alone are sufficient to explain the three-step “immunoediting” concept – the modulation of tumor growth through inhibition, selection and promotion.

Inhibition of COX-2 in Colon Cancer Modulates Tumor Growth and MDR-1 Expression to Enhance Tumor Regression in Therapy-Refractory Cancers In Vivo

Higher cyclooxygenase 2 (COX-2) expression is often observed in aggressive colorectal cancers (CRCs). Here, we attempt to examine the association between COX-2 expression in therapy-refractory CRC, how it affects chemosensitivity, and whether, in primary tumors, it is predictive of clinical outcomes. Our results revealed higher COX-2 expression in chemoresistant CRC cells and tumor xenografts. In vitro, the combination of either aspirin or celecoxib with 5-fluorouracil (5-FU) was capable of improving chemosensitivity in chemorefractory CRC cells, but a synergistic effect with 5-FU could only be demonstrated with celecoxib. To examine the potential clinical significance of these observations, in vivo studies were undertaken, which also showed that the greatest tumor regression was achieved in chemoresistant xenografts after chemotherapy in combination with celecoxib, but not aspirin. We also noted that these chemoresistant tumors with higher COX-2 expression had a more aggressive growth rate. Given the dramatic response to a combination of celecoxib + 5-FU, the possibility that celecoxib may modulate chemosensitivity as a result of its ability to inhibit MDR-1 was examined. In addition, assessment of a tissue microarray consisting of 130 cases of CRCs revealed that, in humans, higher COX-2 expression was associated with poorer survival with a 68% increased risk of mortality, indicating that COX-2 expression is a marker of poor clinical outcome. The findings of this study point to a potential benefit of combining COX-2 inhibitors with current regimens to achieve better response in the treatment of therapy-refractory CRC and in using COX-2 expression as a prognostic marker to help identify individuals who would benefit the greatest from closer follow-up and more aggressive therapy.

Isotropic stress reduces cell proliferation in tumor spheroids

In most instances, tumors have to push their surroundings in order to grow. Thus, during their development, tumors must be able to both exert and sustain mechanical stresses. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spherical cell aggregate. Our results indicate the possibility to modulate tumor growth depending on the applied pressure. Moreover, we demonstrate quantitatively that the cells located in the core of the spheroid display a different response to stress than those in the periphery. We compare the results to a simple numerical model developed for describing the role of mechanics in cancer progression.

Abstract 388: mS100a7a15 enhances mammary tumor growth and metastasis by recruiting tumor associated macrophages

Abstract mS100a7a15 is the murine ortholog of human S100A7 and S100A15 proteins. Both S100A7 and S100A15 have been shown to play an important role in breast cancer. S100A7 has been shown to be highly expressed in high grade ductal carcinoma in situ and invasive breast cancers. Its expression is also related to poor prognosis and associated with increased inflammatory infiltrates and various inflammatory disorders. However, the exact mechanism by which S100A7 or S100A15 enhances breast cancer growth is not known. In the present study, we determined the molecular mechanisms by which mS100a7a15 enhances growth by overexpressing mS100a7a15 in MDA-MB-231 cells. We showed that mS100a7a15 enhances expression of proinflammatory molecules CXCL1 and CXCL8. In addition, we observed that supernatants obtained from mS100a7a15 overexpressing cells enhanced chemotaxis of murine RAW264.7 macrophages. Further elucidation revealed that mS100a7/a15 mediates its effects by binding to receptor for advanced glycation end products (RAGE). We further analyzed the role of mS100a7a15 on modulation of tumor growth and inflammatory pathways in breast cancer by generating inducible bi-transgenic MMTV-rtTA; mS100a7a15 mice (MMTV-mS100a7a15). These mice showed enhanced mS100a7a15 protein expression upon doxycycline treatment. Mammary glands isolated from these mice showed enhanced hyperplasia upon doxycycline treatment for 3 months compared to uninduced mice. Further studies revealed enhanced recruitment of macrophages in mammary glands and activation of STAT3 in induced mice. Orthotopic implantation of MVT-1 breast tumor cells (derived from MMTV-c-Myc; MMTV-VEGF mice) into the mammary glands of these mice showed enhanced tumor growth and metastasis in doxycycline treated mice compared to the control. Tumors and lung tissues obtained from these mice showed enhanced pro-metastatic gene expression and recruitment of F4/80 and CD206 positive M2 tumor-associated macrophages (TAM). However, no difference was observed in CD3+ and CD4+ T lymphocytes. Further elucidation of the role of macrophages by in vivo depletion of macrophages using clodronate liposomes revealed that mS100a7a15-mediated recruitment of TAM is important for tumor growth, angiogenesis and metastasis. Furthermore, mice treated with STAT3 inhibitors showed reduced mS100a7a15-induced hyperplasia. STAT3 has been shown to regulate expression of various inflammatory molecules. These studies using a novel mS100a7a15 bi-transgenic model system demonstrate that mS100a7a15 enhances breast tumor growth and metastasis by enhancing inflammatory signals that result in enhanced recruitment of TAM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 388. doi:1538-7445.AM2012-388

Abstract 4736: N-myc downstream regulated gene 1: A novel mediator of hypoxia-driven chemoresistance in glioblastoma

Abstract Like other solid tumors, glioblastomas (GB) feature vast hypoxic areas that are thought to promote tumor growth, angiogenesis and even drug resistance and hence facilitate an aggressive growth and recurrence after treatment. Elucidation of hypoxia-signalling and identification of the major regulators of hypoxia-dependent chemoprotection seems logical for the optimization of current treatment regimens. Using comparative 2D gel electrophoresis and mass spectrometry analysis, we here identify N-myc downstream regulated gene 1 (NDRG1) as upregulated by chronic sublethal hypoxia in GB cell lines. Immunohistochemical analysis of human astrocytoma specimens shows a marked increase in expression from WHO °II to °IV and a preferential perinecrotic localization of NDRG1 in GB. In vitro, besides the hypoxia-driven induction of NDRG1, which is mediated by Hif1α and Hif2α, NDRG1 is also induced by irradiation. NDRG1 limits glioma cell invasiveness in vitro and reduces proliferation in vitro and in vivo. Furthermore NDRG1 acts antiangiogenic with reduced endothelial cell migration and sprouting, and interferes with the expression of multiple angiogenesis-related signalling molecules. Most interestingly NDRG1 protects from temozolomide (TMZ)-induced G2/M-arrest and subsequent reduction of tumor cell proliferation. Analyzing pairs of GB tissue samples of relapsed glioblastoma patients reveals a dramatic increase of NDRG1-positive cells suggesting a treatment-related selection for NDRG1 expressing populations and a potential role for NDRG1 in the recurrence of GB. In line with these findings, quantifying the NDRG1 status in tissue specimens of the UKT05 GB trial, which looked at a dose-intensified TMZ therapy plus standard radiotherapy, reveals a negative correlation between NDRG1 status and overall survival. In the present work we identify hypoxia-driven NDRG1 as a modulator of tumor growth, invasion, angiogenesis and as a novel mediator of chemoresistance towards TMZ in GB. Our findings shed light on the role of hypoxia in adaption to chemotherapeutic treatment and introduce NDRG1 as a promising candidate for targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4736. doi:1538-7445.AM2012-4736

Leptin Increases HER2 Stability through HSP90 in Breast Cancer Cells

High serum levels of the adipocyte‐derived hormone leptin in obesity are a risk for breast cancer in postmenopausal women. The HER2/Neu receptor, overexpressed in 25% of human breast cancers, has been associated with endocrine resistance.Here, we evaluated the link between leptin and HER2 in human MCF‐7 breast cancer cells and in MCF‐7/HER2‐18 cells stably overexpressing HER2.We found that leptin treatment increased HER2 protein levels, without affecting HER2 mRNA levels. Leptin enhanced the expression of HSP90, a chaperone protein that regulates HER2 degradation, and increased HER2/HSP90 interaction. These events were dependent on JAK2/STAT3 activation, since inhibition of this cascade by AG490 or a STAT3 dominant negative abrogated leptin‐induced HER2 expression. Moreover, leptin treatment increased EGF‐mediated HER2, AKT and MAPK phosphorylation. Anchorage‐independent growth assays revealed that long term leptin treatment reduced sensitivity to the antiestrogen tamoxifen, while increased responsiveness to herceptin, a monoclonal antibody against HER2.We demonstrate that leptin, through HSP90, enhances HER2 stability, providing further insights into the crucial role of leptin in modulating tumor growth and aggressiveness. Clinically, our results suggest that leptin and HSP90 might be novel targets for therapeutic intervention in breast cancer, especially in obese patients.

Abstract A60: Germline genetic variation modulates tumor progression and metastasis in mouse prostate neuroendocrine carcinoma

Abstract Conventional assessment of prognosis in prostate cancer (PC) relies heavily upon Gleason grading, which suffers from significant interobserver variability. Consequently, there is a need for additional means of assessing prognosis. Neuroendocrine differentiation (NED) has been gaining increasing attention as a potential prognostic marker. Pure NE PC, which is associated with a poor outcome, is rare and comprises <1% of all tumors. Additionally, conventional prostate adenocarcinomas displaying NED are associated with a poorer prognosis, with NED increased in high-grade and high-stage tumors, and particularly in hormonally treated and castration resistant disease. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NE PC. We crossed TRAMP mice to the eight progenitor strains of the Collaborative Cross (CC) recombinant inbred (RI) panel to address this. Transgene-positive F1 males were aged to whichever of the following were achieved first: 30 weeks of age or humane endpoints. Tumor growth and metastasis burden were quantified in these mice. These experiments conclusively demonstrate that the introduction of germline variation by breeding significantly modulates tumor growth, local metastasis burden and distant metastasis frequency in this model of NE PC. Compared to wildtype TRAMP males (TRAMP × C57BL/B6J F1; av. tumor burden = 1.40g, n=35) both TRAMP × NOD/ShiLtJ (n=42) and TRAMP × NZO/HlLtJ (n=57) F1 males displayed a significant increase in tumor growth (av. tumor burden = 8.01g and 4.71g, respectively; p<0.0001). Conversely, both TRAMP × WSB/EiJ (n=68) and TRAMP × PWK/PhJ (n=40) F1 males displayed a significant reduction in tumor growth (av. tumor burden = 0.71g and 0.01g, respectively; p<0.0001). Strikingly, only 6 of 40 TRAMP × PWK/PhJ males developed macroscopic tumors, which implies that polymorphisms carried by this strain play a significant role in NED. In those mice with local dissemination, nodal metastasis burden was quantified by normalizing nodal metastasis weight to total prostate tumor weight. In spite of TRAMP × NZO/HlLtJ F1 males (n=41) having an increased tumor burden compared to wildtype TRAMP mice (n=25), a less metastatic phenotype was evident in this strain with normalized metastasis burden being reduced (met burden = 0.23g/g tumor vs. 0.40g/g tumor in wildtype TRAMP mice; p=0.002). Conversely, TRAMP × WSB/EiJ males (n=14), which displayed a reduced tumor burden, had an increased nodal metastasis burden (met burden = 3.20g/g tumor; p=0.009). Patterns of distant metastasis to lung tended to follow the same patterns as for local dissemination in each of the strains. Tumor-associated morbidity was also significantly higher in four F1 strains (A/J, CAST/EiJ, NOD/LtJ and NZO/HlLtJ) compared to control mice, implying that the excess tumor burden in these strains has a deleterious effect upon survival. All tumors and metastases displayed positive staining for NE markers, and quantification of the expression of these is ongoing to determine strain-specific differences. The eventual aim of this work is to identify quantitative trait loci associated with the development of NED and aggressive disease development in PC. We are therefore performing F2 intercrosses between TRAMP mice and a number of the strains described above. Additionally, TRAMP mice are being bred to Diversity Outbred stock, which is a genetically diverse mouse resource used as a tool for high resolution genetic mapping. Candidate gene characterization within these loci will facilitate an improved understanding of this aspect of PC pathogenesis and potentially facilitate the identification of individuals at increased risk of NED. Citation Format: Shashank Patel, Erin Kollins, Katie Bell, Robert Cardiff, Alfredo Molinolo, Nigel Crawford. Germline genetic variation modulates tumor progression and metastasis in mouse prostate neuroendocrine carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A60.

Stress Clamp Experiments on Multicellular Tumor Spheroids

Cancer progress is a multistep process. In contrast with the genetic aberrations, the precise role of the micro-environment and its interplay with the tumor is still poorly understood. In this work, we present two different approaches to measure the effect of mechanical stress on tissue growth and death. The first setup use osmotic pressure to deform a dialysis bag and exert a known pressure on a multicellular tumor spheroid. Our results indicate the ability to modulate tumor growth depending on the applied pressure. Moreover, we demonstrate quantitatively that cells have a different response to stress whether they are located in the core or in the periphery of the spheroid. The second setup is a microfluidic based integrated system which enables to feed and visualize spheroids in the same time that we apply a known pressure. We compare the results to numerical and analytic models developed to describe the role of mechanics in cancer progression. Fabien Montel, Morgan Delarue, Jens Elgeti, Laurent Malaquin, Markus Basan, Thomas Risler, Bernard Cabane, Danijela Vignjevic, Jacques Prost, Giovanni Cappello and Jean-François Joanny. Stress clamp experiments on multicellular tumor spheroids. Physical Review Letters. In press.