Aggressive Behavior Of Tumor Cells Research Articles

Abstract 2510: Pentraxin-3 associated up-regulation of genes involved in motility, angiogenesis, and inflammation in glioblastoma

Abstract Glioblastoma, a malignant brain cancer, is highly invasive and resistant to multimodal treatment partly due to distorted vasculature and exacerbated inflammation in its tumor microenvironment. The glycoprotein pentraxin-3 (PTX3), a modulator of inflammation, is correlated with the severity of some cancers. However, the mechanism responsible for the oncogenic role of PTX3 in GBM malignancy remains unclear. Our previous study examined the role of PTX3 in GBM growth, angiogenesis, and invasion using in vitro glioblastoma cells and in vivo/pre-clinical GBM models, proteomic profiling, molecular and biochemical approaches. Over-expression of PTX3 in U87 brain tumor cells correlated with cell cycle progression, increased migratory potential, and survival under hypoxic conditions. PTX3 over-expression increased blood vessel forming ability of endothelial cells. Importantly, PTX3 over-expression increased tumor growth, micro-vessel density, and invasion in brains of an orthotopic mouse model. To understand the molecular mechanisms associated with PTX3 mediated tumor aggressiveness, we further examined the effects of PTX3 on mRNA levels of genes involved in motility/invasion, angiogenesis, and inflammation using pathway specific gene arrays. PTX3 over expression was associated with the 5-fold upregulation of hypoxia-inducible factor (HIF)-1α, which in turn transactivates several genes essential for tumor growth and survival. Indeed, higher PTX3 expression led to increased mRNA levels of genes involved in motility, angiogenesis, and inflammation. The upregulated motility associated genes included ARHGEF7, RAC1, and PAK, a protein kinase involved in intracellular signaling pathways downstream of integrins. Inflammatory genes that were increased in expression included IL-1beta, CCL3, CCL5, and CXCL5 that amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway. The upregulated pro-angiogenic genes included interleukin-8 (IL-8), matrix metalloproteinase-2 (MMP2), and vascular endothelial growth factor (VEGF), expression of which correlates with GBM malignancy. Interestingly PTX3 decreased the levels of thrombospondin-1 that inhibits angiogenesis. Thus, PTX3 appears to trigger multiple signaling pathways that enhance the aggressive behavior of tumor cells. Taken together, our study indicates that targeted therapy such as blocking PTX3 may reduce the aggressiveness of glioblastomas through regulation of multiple molecular pathways. Citation Format: Umadevi V. Wesley, Ian Sutton, Dylan E. Nelson, Paul A. Clark, Robert J. Dempsey. Pentraxin-3 associated up-regulation of genes involved in motility, angiogenesis, and inflammation in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2510.

In Vitro Evidence of Differential Immunoregulatory Response between MDA-MB-231 and BT-474 Breast Cancer Cells Induced by Bone Marrow-Derived Mesenchymal Stromal Cells Conditioned Medium.

Inside tumors, cancer cells display several mechanisms to create an immunosuppressive environment. On the other hand, by migration processes, mesenchymal stromal cells (MSCs) can be recruited by different cancer tumor types from tissues as distant as bone marrow and contribute to tumor pathogenesis. However, the impact of the immunoregulatory role of MSCs associated with the aggressiveness of breast cancer cells by soluble molecules has not been fully elucidated. Therefore, this in vitro work aimed to study the effect of the conditioned medium of human bone marrow-derived-MSCs (hBM-MSC-cm) on the immunoregulatory capability of MDA-MB-231 and BT-474 breast cancer cells. The hBM-MSC-cm on MDA-MB-231 cells induced the overexpression of TGF-β, IDO, and IL-10 genes. Additionally, immunoregulation assays of mononuclear cells (MNCs) in co-culture with MDA-MB-231 and hBM-MSC-cm decreased lymphocyte proliferation, and increased proteins IL-10, TGF-β, and IDO while also reducing TNF levels, shooting the proportion of regulatory T cells. Conversely, the hBM-MSC-cm did not affect the immunomodulatory capacity of BT-474 cells. Thus, a differential immunoregulatory effect was observed between both representative breast cancer cell lines from different origins. Thus, understanding the immune response in a broader tumor context could help to design therapeutic strategies based on the aggressive behavior of tumor cells.

ITGB1 Drives Hepatocellular Carcinoma Progression by Modulating Cell Cycle Process Through PXN/YWHAZ/AKT Pathways.

Integrin β1 (ITGB1), which acts as an extracellular matrix (ECM) receptor, has gained increasing attention as a therapeutic target for the treatment of hepatocellular carcinoma (HCC). However, the underpinning mechanism of how ITGB1 drives HCC progression remains elusive. In this study, we first found that ITGB1 expression was significantly higher in HCC tissues than in normal controls by bioinformatics analysis. Furthermore, bioinformatics analysis revealed that paxillin (PXN) and 14-3-3 protein zeta (YWHAZ) are the molecules participating in ITGB1-regulated HCC tumor cell cycle progression. Indeed, immunohistochemistry (IHC) revealed that ITGB1, paxillin, and YWHAZ were strongly upregulated in paired HCC tissue compared with adjacent normal tissues. Notably, the inhibition of ITGB1 expression by small interfering RNA (siRNA) resulted in the downregulated expression of PXN and YWHAZ in primary HCC cells, as assessed by western blot and immunostaining. In addition, ITGB1 knockdown markedly impaired the aggressive behavior of HCC tumor cells and delayed cell cycle progression as determined by cell migration assay, drug-resistance analysis, colony formation assay, quantitative real-time polymerase chain reaction (qRT-PCR), and cell cycle analysis as well as cell viability measurements. More importantly, we proved that xenograft ITGB1high tumors grew more rapidly than ITGB1low tumors. Altogether, our study showed that the ITGB1/PXN/YWHAZ/protein kinase B (AKT) axis enhances HCC progression by accelerating the cell cycle process, which offers a promising approach to halt HCC tumor growth.

A glutaminase isoform switch drives therapeutic resistance and disease progression of prostate cancer

Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to hormonal therapy resistance and disease progression in prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) up-regulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), an androgen-independent GLS1 isoform with more potent enzymatic activity, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyperproliferation, and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GAC shows better treatment effect for castration-resistant PCa than for hormone-sensitive PCa in vitro and in vivo. In summary, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is one of the key mechanisms in therapeutic resistance and disease progression.

A Glutaminase Isoform Switch Drives Therapeutic Resistance and Disease Progression of Prostate Cancer

Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to the hormonal therapy resistance and disease progression of prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) upregulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), a shorter splicing isoform of GLS1that is upregulated by MYC (c-MYC and N-Myc) with more potent in glutaminolysis, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyper-proliferation and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GLS1 isoforms differentially suppresses the growth of hormone sensitive prostate cancer and castration resistant prostate cancer in vitro and in vivo. Therefore, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is a key mechanism in therapeutic resistance and disease progression.

Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8

BackgroundThe Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer.MethodsWe used RNA silencing to decrease levels of expression in mouse mammary tumor cells and overexpression of ZC3H8 in cells derived from the normal mouse mammary gland. We measured characteristics of cell behavior in vitro, including proliferation, migration, invasion, growth in soft agar, and spheroid growth. We assessed the ability of these cells to form tumors in syngeneic BALB/c mice. ZC3H8 protein was visualized in cells using confocal microscopy.ResultsTumor cells with lower ZC3H8 expression exhibited decreased proliferation rates, slower migration, reduced ability to invade through a basement membrane, and decreased anchorage independent growth in vitro. Cells with lower ZC3H8 levels formed fewer and smaller tumors in animals. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells led to an opposite effect. ZC3H8 protein localized to both PML bodies and Cajal bodies within the nucleus. ZC3H8 has a casein kinase 2 (CK2) phosphorylation site near the N-terminus, and a CK2 inhibitor caused the numerous PML bodies and ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restored PML bodies to their original state. A mutant ZC3H8 lacking the predicted CK2 phosphorylation site showed localization and numbers of ZC3H8/PML bodies similar to wild type. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable threonine showed dramatically increased numbers of smaller nuclear foci.ConclusionsThese experiments demonstrate that Zc3h8 expression contributes to aggressive tumor cell behavior in vitro and in vivo. Our studies show that ZC3H8 integrity is key to maintenance of PML bodies. The work provides a link between the Little Elongation Complex, PML bodies, and the cancer cell phenotype.

Abstract 2484: Structural analysis of features contributing to the oncogenic phenotype conferred by the zinc finger nuclear body protein Zc3h8

Abstract The zinc finger protein Zc3h8 is overexpressed in many mouse and human solid tumors. We have shown that increased expression of Zc3h8 in cultured mouse mammary cells is associated with increased proliferation rate, increased motility and invasiveness, increased capacity for growth in three dimensions, and increased capacity for tumor formation. Partial knockdown of Zc3h8 expression from mouse mammary tumor cells has the opposite effect. The Zc3h8 protein localizes to both PML bodies and Cajal bodies in the nucleus and features of this localization are altered by treatment of cells with a casein kinase 2 inhibitor, TBB. We have embarked upon an analysis of the contributions of structural features of the Zc3h8 protein through mutagenesis of the gene at key positions. The casein kinase 2 phosphorylation site at position T32 was mutated to either an alanine to prevent phosphorylation, or to glutamic acid to mimic constitutive phosphorylation. Each of the three zinc fingers in the C-terminal region was mutated to replace one cysteine residue. The mutations were introduced into a variant of the zc3h8 gene that has 6 mutations introduced at synonymous positions in order to escape knockdown by RNAi, although the protein sequence generated is identical to the original Zc3h8. Each of the mutant constructs was introduced into mouse mammary tumor cells with knockdown of the endogenous Zc3h8. Mutation of the phosphorylation site T32A led to the formation of fewer, although larger PML bodies, with continued ZC3h8 co-localization. The T32E mutant, with constitutive negative charge at this position led to the formation of a large number of small nuclear foci. Mutant constructs were stably transfected into tumor cells with knockdown of endogenous Zc3h8, and behavior of the mutants was compared to the behavior of cells transfected with the synonymously mutated gene construct. Disruption of each of the zinc fingers individually led to decreased proliferation rate, decreased growth as spheroids or in soft agar, and decreased invasion through Matrigel relative to the synonymous Zc3h8. Tumor cells with Zc3h8 knockdown failed to form tumors in syngeneic BALB/c mice within 10 weeks, while cells transfected with a negative control vector formed tumors in all animals. Rescue of Zc3h8 expression with the synonymous mutant led to tumor formation in 50% of the animals, while no tumors formed from cells transfected with zinc finger mutants. We conclude that the zinc fingers contribute to aggressive tumor cell behavior while the T32 phosphorylation site is essential for proper localization to nuclear bodies. Citation Format: John A. Schmidt, Emily R. Duffner, Emily M. Harris, Tyler Doan, Emanuel Irizarry, Keith G. Danielson, Janice E. Knepper. Structural analysis of features contributing to the oncogenic phenotype conferred by the zinc finger nuclear body protein Zc3h8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2484.

Distinct Activities of Glycolytic Enzymes Identify Chronic Lymphocytic Leukemia Patients with a more Aggressive Course and Resistance to Chemo-Immunotherapy

A higher capacity to grow under hypoxic conditions can lead to a more aggressive behavior of tumor cells. Determining tumor activity under hypoxia may identify chronic lymphocytic leukemia (CLL) with aggressive clinical course and predict response to chemo-immunotherapy (CIT). A metabolic score was generated by determining pyruvate kinase and lactate dehydrogenase, key enzymes of glycolysis, ex vivo in primary CLL samples under normoxic and hypoxic conditions. This score was further correlated with clinical endpoints and response to CIT in 96 CLL patients. 45 patients were classified as metabolic high risk (HR), 51 as low risk (LR). Treatment-free survival (TFS) was significantly shorter in HR patients (median 394 vs 723 days, p = .021). 15 HR patients and 14 LR patients received CIT after sample acquisition. HR patients had a significantly shorter progression-free survival after treatment compared to LR patients (median 216 days vs not reached, p = .008). Multivariate analysis evaluating age, IGHV, TP53 deletion or mutation and 11q22–23 deletion besides the capacity of tumor cells to grow under severe hypoxic conditions identified the metabolic profile as the strongest independent risk factor for shorter TFS (hazard ratio 2.37, p = .011). The metabolic risk can provide prognostic and predictive information complementary to genetic biomarkers and identify patients who might benefit from alternative treatment approaches.

Macrophages induce "budding" in aggressive human colon cancer subtypes by protease-mediated disruption of tight junctions.

Primary human colorectal tumors with a high stromal content have an increased capacity to metastasize. Cancer-associated fibroblasts (CAFs) promote metastasis, but the contribution of other stromal cell types is unclear. Here we searched for additional stromal cell types that contribute to aggressive tumor cell behavior. By making use of the ‘immunome compendium’—a collection of gene signatures reflecting the presence of specific immune cell-types—we show that macrophage signatures are most strongly associated with a high CAF content and with poor prognosis in multiple large cohorts of primary tumors and liver metastases. Co-culturing macrophages with patient-derived colonospheres promoted ‘budding’ of small clusters of tumor cells from the bulk. Immunohistochemistry showed that budding tumor clusters in stroma-rich areas of T1 colorectal carcinomas were surrounded by macrophages. In vitro budding was accompanied by reduced levels of the tight junction protein occludin, but OCLN mRNA levels did not change, nor did markers of epithelial mesenchymal transition. Budding was accompanied by nuclear accumulation of β-catenin, which was also observed in budding tumor cell clusters in situ. The NFκB inhibitor Sanguinarine resulted in a decrease in MMP7 protein expression and both NFκB inhibitor Sanguinarine and MMP inhibitor Batimastat prevented occludin degradation and budding.We conclude that macrophages contribute to the aggressive nature of stroma-rich colon tumors by promoting an MMP-dependent pathway that operates in parallel to classical EMT and leads to tight junction disruption.

Abstract 4805: The role of exosomes in fibrosarcoma progression

Abstract Among pediatric cancers, sarcomas, especially those with large tumor burdens and metastatic disease, often result in poor outcome. Thus, new treatments are urgently needed to inhibit tumor progression, prevent metastasis, and improve overall survival. To understand the mechanisms driving sarcoma progression, we employed two mouse fibrosarcoma cell lines that display different growth phenotypes when transplanted into syngeneic immune competent mice. Progressor fibrosarcomas evade detection by the immune system and develop large tumor burdens, while regressor fibrosarcomas regress shortly after a period of limited tumor growth. This difference in the growth phenotype is mediated in part by immune cells, but the mechanisms by which progressor and regressor cells influence immune cell activity are not fully elucidated. Our research has focused on exosomes, 50-100 nm secreted nanovesicles, that contain bioactive cargoes and have emerged as mediators of intercellular communication between various cells. Here, we hypothesize that fibrosarcoma exosomes determine the aggressiveness of the disease by either educating tumor cells themselves or altering interactions with host cells. To investigate the role of fibrosarcoma exosomes, we first educated regressor tumor cells with progressor exosomes for 3 weeks in vitro, and vice versa. Upon in vivo inoculation, progressor cells conditioned with regressor cell-derived exosomes grew significantly slower compared to untreated progressor cells. In contrast, regressor cells educated with progressor exosomes did not regress and overgrew significantly compared to untreated regressor cells. These results indicate that exosomes have an ability to reprogram a tumor phenotype. To determine if fibrosarcoma exosomes specifically impact the host, we evaluated whether conditioning naïve mice with progressor exosomes alters in vivo growth of regressor tumor cells injected after three weeks of education. Twenty percent of regressor tumors continued to grow following progressor exosome education, while all tumors regressed in the control PBS group. These data suggest that exosomes also play an important role in the interaction with the host cells influencing tumor growth and disease progression. Next, to determine which exosomal cargo may mediate these effects, we sought to characterize the proteome of exosomes from progressor and regressor cells using mass spectrometry. We found basigin, also known as CD147, a transmembrane embryonic glycoprotein reported to regulate cell proliferation, tumor migration and metastasis as one of the candidate proteins highly expressed in progressor-derived exosomes. We conclude that exosomes derived from fibrosarcoma progresssors and regressors can alter the tumor phenotype both directly and indirectly. Exosomal cargo, such as basigin, may mediate the aggressive behavior of tumor cells, making it a potential therapeutic target to inhibit fibrosarcoma progression. Citation Format: Miho Nakajima, Stephen C. Searles, Ayuko Hoshino, Katherine M. Offer, Candia M. Kenific, Jack D. Bui, David C. Lyden. The role of exosomes in fibrosarcoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4805. doi:10.1158/1538-7445.AM2017-4805

Abstract 2555: ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior

Abstract The ZC3H8 gene encodes a protein with three zinc finger motifs in the C-terminal region, suggesting a role as an RNA binding protein, but whose specific function remains unclear. ZC3H8 protein localizes to both PML bodies and Cajal bodies within the nucleus, as confocal microscopy demonstrates colocalization of ZC3H8 with both PML and coilin proteins. ZC3H8 has a casein kinase 2 phosphorylation site in the N-terminal region, and treatment with a casein kinase 2 inhibitor causes the numerous PML bodies and their associated ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restores PML bodies to their original state. We constructed a mutant of ZC3H8 that removed the predicted CK2 phosphorylation site by replacing the serine with an alanine residue. Cells transfected with this mutant had reduced numbers of PML bodies, similar to cells treated with the CK2 inhibitor. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable serine was predicted to behave as a constitutive mutant. This mutant did not exhibit differences from wild type localization. These experiments suggest that ZC3H8 integrity is key to maintenance of PML bodies. Other studies have identified ZC3H8 as a component of the LEC (little elongation complex), and we found that another identified component, ICE2 (Narg) also localizes to PML bodies. The ZC3H8 gene is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. We have used RNA silencing to decrease levels of expression in two independent mouse mammary tumor cell lines. Cells with lower ZC3H8 expression have dramatic phenotypic changes, showing decreased rates of proliferation, slower migration in wound healing assays, formation of fewer and smaller colonies in soft agar assays, and decreased ability to invade through a basement membrane in transwell assays. Further, ZC3H8-depleted cells have decreased rates of growth as spheroids and decreased growth in vivo in syngeneic mice. We developed an inducible vector driving expression of ZC3H8. Upon treatment with the inducer, doxycycline, cells assume a more aggressive phenotype. We introduced a synonymous mutant of ZC3H8 unable to be silenced by the RNAi into the knockdown cells, and rescued the aggressive phenotype of the original tumor cells. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells leads to a less pronounced, but opposite, increase in proliferation, motility, and invasion. We have engineered the synonymous ZC3H8 with disrupted zinc finger motifs, and preliminary results suggest that disruption of the first zinc finger is not sufficient to cause loss of the aggressive phenotype. We suggest that ZC3H8 is an oncogene with substantial effects on the cancer cell phenotype. These effects are hypothesized to be associated with ZC3H8 function in PML bodies. Citation Format: John A. Schmidt, Emily Duffner, Gerard Walker, Keith G. Danielson, Janice E. Knepper. ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2017-2555

A case report of primary cutaneous natural killer/T-cell lymphoma.

Primary cutaneous extranodal natural killer/T-cell lymphoma, nasal type (ENKL-NT) is a relatively rare disease associated with aggressive tumor-cell behavior and poor prognosis. Progress in immunohistochemistry has improved the identification of ENKL-NT. The present case study reported on a 64-year-old female patient presenting with several red nodular lesions on the neck developed over four months. Cutaneous biopsy revealed these cells were positive for CD3, CD56, CD5, CD8 and negative for CD2, CD34, CD7, CD20 and Granzyme B. A computed tomography scan and bone marrow biopsy did not show any abnormalities and a diagnosis of primary cutaneous ENKL-NT was made. After treatment with chemotherapy regimens comprising cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), followed by high-dose treatment with methotrexate, dexamethasone, ifosfamide, etoposide and L-asparaginasum (SMILE), the patient succumbed to the disease. The present study exemplified that immunohistochemical analysis as well as the recognition of atypical lymphoid cells showing angiocentricity is crucial for the correct diagnosis of ENKL-NT. The prognosis of primary cutaneous ENKL-NT remains poor. As the CHOP regimen is not completely effective, high-dose chemotherapy, such as the SMILE regimen, is required.

Diethyldithiophosphate (DEDTP) Role in Proportion of Macrophages and Natural Killer Cells in Lymph Vessel and 4T1 Murine Breast Tumor Model

The diethyldithiophosphate (DEDTP) is a metabolite produced by the degradation of organophosphorus pesticides (POFs), and a dialkylphosphate (DAP) which is also chemically synthesized with a widespread commercial use and so the world population is chronically exposed to this compound. It is suggested that organophosphorus compounds produce deleterious effects on the immune system of the exposed population, since it has been demonstrated that DAPs affect peripheral blood lymphocytes activation and proliferation of T lymphocytes, furthermore, in vivo assays using C57 mice, the exposure to DEDTP enlarges skin tumors of BMK (Baby Mouse Kidney) cells, these results suggest that DEDTP exposure in vivo may modify the immune response and enhance the growth of tumor cells. Therefore, our objective is to determine the effect of DEDTP in the proportion of macrophages and natural killer cells (NKs) in lymph nodes and 4T1 breast cancer tumors in mice. To this end, we used Balb/c female mice of 6–8 weeks old, exposed for 8 days to 0.01 g/kg i.p. of DEDTP and inoculated with 1×105 4T1 cells. Tumors and lymph nodes were excised and measured. The percentage of total (F4/80+) and M2 (CD206+) macrophages and natural killer (NK) cells (CD355+) were assessed by flow cytometry. Exposure to DEDTP for 8 days increased the proportion of M2 macrophages. After 15 days of exposure to DEDTP, NK cells proportion was reduced but increased after 20 days. In mice challenged with 4T1 cells during DEDTP exposure, the number of total macrophages increased while the proportion of CD355+ cells in the lymph node proximal to the tumor decreased after 5 days; 10 days after, the number of M2 macrophages increased. Finally, after 20 days the number of M2 macrophages and NK cells in the proximal lymph node increased. In tumors, the number of infiltrating NK cells and total macrophages were reduced, followed by a reduction of M2 macrophages. Subsequently, after 15 days we observed an increase of infiltrating NK cells and M2 macrophages; after 20 days of tumor, M2 macrophages were increased. None of these changes had a significant impact on the establishment and development of tumors. With these results we conclude that exposure to DEDTP, in the absence of a particular antigen challenge, exerts immunomodulatory effects on individuals, apparently by inducing alternative activation of macrophages, however, this effect had no significant effect in the growth of 4T1 tumor cells. This could probably be because the more aggressive behavior of 4T1 tumor cells than BMK cells (previously tested on C57 mice) and the pronounced Th2 immune response profile of Balb/c mice (contrary to C57 animals). These results suggest that the effect of DEDTP on the immune system is not decisive against an aggressive tumor challenge, but may have relevance against a bacterial or parasitic infectious challenge.Support or Funding InformationCONACyT grant 153468

Abstract 3945: PTEN loss is associated with aggressive behavior and poor prognosis in Middle Eastern Triple Negative Breast Cancer

Abstract PTEN is a tumor suppressor that negatively regulates the PI3K-AKT signaling pathway which is involved in the pathogenesis of many different tumor types and serves as a prognostic marker in breast cancer. However, the significance of the role of PTEN in Middle Eastern ethnic breast cancer has not been explored especially with the fact that breast cancer originating from this ethnic population tend to behave more aggressively than breast cancer in the west. In order to learn more on the prevalence and clinical significance of PTEN protein expression and PTEN gene deletions, a tissue microarray containing more than 1000 primary breast cancers with clinical follow up data was used. Tissue Microarray sections were analyzed for protein expression and copy number change using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Loss of PTEN immunostaining was observed in 77% of the cases. PTEN loss was significantly associated with large tumor size (p = 0.0030), high grade (p = 0.0281), tumor recurrence (p = 0.0333) and Triple negative breast cancers (p = 0.0086). PTEN loss in Triple negative breast cancers was significantly associated with rapid tumor cell proliferation (p = 0.0396) and poor prognosis (p = 0.0408). PTEN deletion was found only in 60 cases (6.4%) of cases. It was concluded that loss of PTEN protein expression occurs at high frequency in Middle Eastern breast cancer. PTEN inactivation may potentially lead to an aggressive behavior of tumor cells through stimulation of tumor cell proliferation. Furthermore PTEN signaling pathway might be used as potential therapeutic target in Triple negative breast cancers since loss of its expression is shown to be significantly associated with this aggressive subtype of breast cancer. Citation Format: Abdul K. Siraj, Shaham Beg, Sarita Prabhakaran, Zeenath Jehan, Dahish Ajarim, Fouad Al-Dayel, Asma Tulbah, Khawla Al-Kuraya. PTEN loss is associated with aggressive behavior and poor prognosis in Middle Eastern Triple Negative Breast 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 3945. doi:10.1158/1538-7445.AM2015-3945

Loss of PTEN expression is associated with aggressive behavior and poor prognosis in Middle Eastern triple-negative breast cancer.

PTEN is a tumor suppressor that negatively regulates the PI3K-AKT signaling pathway which is involved in the pathogenesis of many different tumor types and serves as a prognostic marker in breast cancer. However, the significance of the role of PTEN in Middle Eastern ethnic breast cancer has not been explored especially with the fact that breast cancer originating from this ethnic population tend to behave more aggressively than breast cancer in the west. In this study, we analyzed PTEN alteration in a tissue microarray format containing more than 1000 primary breast cancers with clinical follow-up data. Tissue Microarray sections were analyzed for protein expression and copy number change using immunohistochemistry and fluorescence in situ hybridization. Loss of PTEN immunostaining was observed in 77% of the cases. PTEN loss was significantly associated with large tumor size (p=0.0030), high grade (p=0.0281), tumor recurrence (p=0.0333), and triple-negative breast cancers (p=0.0086). PTEN loss in triple-negative breast cancers was significantly associated with rapid tumor cell proliferation (p=0.0396) and poor prognosis (p=0.0408). PTEN deletion was found only in 60 cases (6.4%). Loss of PTEN protein expression occurs at high frequency in Middle Eastern breast cancer. PTEN inactivation may potentially lead to an aggressive behavior of tumor cells through stimulation of tumor cell proliferation. Furthermore, PTEN signaling pathway might be used as potential therapeutic target in triple-negative breast cancers since loss of its expression is shown to be significantly associated with this aggressive subtype of breast cancer.

Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking

Hyaluronan (HA) turnover accelerates metastatic progression of prostate cancer in part by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed hyaluronidase 1 (Hyal1) as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of HA and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific colocalization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found that enzymatic activity of Hyal1 was necessary for normal localization within the cell as Hyal1-E131Q was mainly detected within the endoplasmic reticulum. Expression of a HA-binding point mutant, Hyal1-Y202F, revealed that secretion of Hyal1 and concurrent reuptake from the extracellular space are critical for rapid HA internalization and cell proliferation. Overall, excess Hyal1 secretion accelerates endocytic vesicle trafficking in a substrate-dependent manner, promoting aggressive tumor cell behavior.

63IN - Targeting Tumour Addiction

ABSTRACT Oncogene addiction refers to dependence of malignancy on a single molecular aberration that drives proliferation and aggressive behavior of tumor cells. In the past decade, deeper understanding of driving molecular aberrations in lung adenocarcinoma led to clinical development of targeted therapies with substantial survival prolongation in respective cohorts of patients. Moreover, it is now apparent that these groups of patients have particular biological and clinical characteristics. Mutations in EGFR, HER2, RAS, BRAF as well as rearrangements in ALK, ROS1, RET and gene amplification of MET serve as validated examples of clinically relevant targets in non-small-cell lung cancer in 2014. For these targets, we have several effective agents and we also understand molecular and clinical characteristics of the most common resistance mechanisms with possibility to block these mechanisms with another set of effective therapies, as exemplified by novel generation of EGFR inhibitors. The challenge for the future is how to characterize new relevant molecular targets in dominant oncogenes but also in tumor suppressor oncogenes and how to effectively document the clinical benefit of their blockade. Integration of targeted therapies with cytotoxic agents and immune checkpoint inhibitors is hoped to further increase therapeutic potential of single-agent inhibitor strategies. Disclosure: R. Dziadziuszko: Consultancy and speaker's agreements (compensated) with Roche, Pfizer, Bristol-Myers Squibb, CLOVIS and Boehringer-Ingelheim

The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

MicroRNAs (miRNAs) are a family of small RNA molecules that negatively regulate the expression of protein-coding genes and play critical roles in orchestrating diverse cellular processes. This regulatory mechanism is also exploited by viruses to direct their life cycle and evade the host immune system. Epstein-Barr virus (EBV) is an oncogenic virus that is closely associated with multiple human diseases, including nasopharyngeal carcinoma (NPC), which is a highly metastatic type of tumor and is frequently reported in South Asia. Several viral proteins have been found to promote the migration and invasiveness of NPC cells. However, not all tumor tissues express these viral oncoproteins, suggesting that other mechanisms may contribute to the aggressive behavior of NPC tumor cells. A previous sequencing study by our group revealed that the EBV miRNA miR-BART9 was expressed at high levels in all EBV-positive NPC tissues. In the present study, we used gain- and loss-of-function approaches to investigate the effect of miR-BART9 in EBV-negative and EBV-positive NPC cells. We discovered that miR-BART9 promotes the migration and invasiveness of cultured NPC cells. The promigratory activity observed in vitro was manifested as an enhanced metastatic ability in vivo. Computational analysis revealed that miR-BART9 may target E-cadherin, a membrane protein that is pivotal in preserving cell-cell junctions and the epithelial phenotype. Through biochemical assays and functional rescue analysis, we confirmed that miR-BART9 specifically inhibits E-cadherin to induce a mesenchymal-like phenotype and promote the migration of NPC cells. These results indicated that miR-BART9 is a prometastatic viral miRNA and suggested that high levels of miR-BART9 in EBV-positive NPC cells may contribute to the aggressiveness of tumor cells.

Obesity and Breast Cancer: A Multipartite Connection

Obesity and Breast Cancer: A Multipartite Connection

Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms

Incidence of skeletal metastases and death from prostate cancer greatly increases with age and obesity, conditions which increase marrow adiposity. Bone marrow adipocytes are metabolically active components of bone metastatic niche that modulate the function of neighboring cells; yet the mechanisms of their involvement in tumor behavior in bone have not been explored. In this study, using experimental models of intraosseous tumor growth and diet-induced obesity, we demonstrate the promoting effects of marrow fat on growth and progression of skeletal prostate tumors. We reveal that exposure to lipids supplied by marrow adipocytes induces expression of lipid chaperone FABP4, pro-inflammatory interleukin IL-1β, and oxidative stress protein HMOX-1 in metastatic tumor cells and stimulates their growth and invasiveness. We show that FABP4 is highly overexpressed in prostate skeletal tumors from obese mice and in bone metastasis samples from prostate cancer patients. In addition, we provide results suggestive of bi-directional interaction between FABP4 and PPARγ pathways that may be driving aggressive tumor cell behavior in bone. Together, our data provide evidence for functional relationship between bone marrow adiposity and metastatic prostate cancers and unravel the FABP4/IL-1β axis as a potential therapeutic target for this presently incurable disease.