Early Stages Of Tumor Progression Research Articles

Abstract B35: NR2F1 limits early dissemination and phenotypic plasticity of early-progressed ErbB2+ tumor cells by maintaining luminal differentiation

Abstract Tumor cells derived from MMTV-Neu primary tumors (tumor-derived cells, TDC) are known to be able to complete all steps of the metastatic cascade. However, new evidence shows that cells derived from early stages of tumor progression (before detection of palpable tumors, ErbB2+ early-progressed tumor cells, EPTC) can disseminate and, as disseminated tumor cells (early DTCs), enter a non-proliferative state for prolonged periods at secondary organs. When EPTC are co-cultured with TDC, the tumor sphere formation capacity of the later is increased suggesting a synergistic contribution of early-progressed tumor cells to the growth of TDC. Thus, early DTCs are proposed to contribute to metastasis, but the mechanisms that would allow these cells, considered sessile and with few genetic alterations, to complete all steps of metastasis are unknown. Here we report that the orphan nuclear receptor NR2F1 is downregulated in human pre-malignant and malignant lesions and mouse ErbB2+ EPTC and TDC when compared with normal mammary tissue. In ErBb2+ EPTC further downregulation of NR2F1 activates a motile phenotype and this is coincident with the detection of circulating tumor cells (CTCs) and DTCs in lungs and bone marrow. Knock down of basal NR2F1 levels in ErBb2+ EPTC induced cell motility, loss of laminin-V deposition, β-catenin delocalization from the membrane and dramatic loss of E-cadherin junctions. Interestingly, TWIST, SMAD2, CK14 and ROR1 levels became upregulated upon NR2F1 depletion. This anti-EMT NR2F1-regulated program is ErbB2 oncogen-dependent. These results suggest that NR2F1 expression maintains epithelial identity and suppresses epithelial-mesenchymal transition (EMT), possibly by blocking WNT signaling. Knock down of NR2F1 in ErBb2+ EPTC also enhanced mammosphere formation efficiency and this was accompanied by upregulation of the pluripotency transcription factor NANOG and TWIST. These findings suggest that NR2F1 limits the spreading of ErBb2+ EPTC cells and it promotes a luminal differentiation-associated program by limiting the activation of a stem cell-like program. Our findings provide for the first time evidence that NR2F1 functions in mammary epithelial cells as a suppressor of pluripotency and dissemination during early stages of tumor progression. We propose that therapies that might restore expression of NR2F1 might limit early dissemination and the progression to metastasis of already disseminated tumor cells. Citation Format: Maria Soledad Sosa, Julie Cheung, Julio A. Aguirre-Ghiso. NR2F1 limits early dissemination and phenotypic plasticity of early-progressed ErbB2+ tumor cells by maintaining luminal differentiation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B35.

Dietary Selenium Supplementation Modulates Growth of Brain Metastatic Tumors and Changes the Expression of Adhesion Molecules in Brain Microvessels.

Various dietary agents can modulate tumor invasiveness. The current study explored whether selenoglycoproteins (SeGPs) extracted from selenium-enriched yeast affect tumor cell homing and growth in the brain. Mice were fed diets enriched with specific SeGPs (SeGP40 or SeGP65, 1 mg/kg Se each), glycoproteins (GP40 or GP65, 0.2–0.3 mg/kg Se each) or a control diet (0.2–0.3 mg/kg Se) for 12 weeks. Then, murine Lewis lung carcinoma cells were infused into the brain circulation. Analyses were performed at early (48 h) and late stages (3 weeks) post tumor cell infusion. Imaging of tumor progression in the brain revealed that mice fed SeGP65-enriched diet displayed diminished metastatic tumor growth, fewer extravasating tumor cells and smaller metastatic lesions. While administration of tumor cells resulted in a significant upregulation of adhesion molecules in the early stage of tumor progression, overexpression of VCAM-1 (vascular call adhesion molecule-1) and ALCAM (activated leukocyte cell adhesion molecule) messenger RNA (mRNA) was diminished in SeGP65 supplemented mice. Additionally, mice fed SeGP65 showed decreased expression of acetylated NF-κB p65, 48 h post tumor cell infusion. The results indicate that tumor progression in the brain can be modulated by specific SeGPs. Selenium-containing compounds were more effective than their glycoprotein controls, implicating selenium as a potential negative regulator of metastatic process.

Neural Regulation of Pancreatic Cancer: A Novel Target for Intervention.

The tumor microenvironment is known to play a pivotal role in driving cancer progression and governing response to therapy. This is of significance in pancreatic cancer where the unique pancreatic tumor microenvironment, characterized by its pronounced desmoplasia and fibrosis, drives early stages of tumor progression and dissemination, and contributes to its associated low survival rates. Several molecular factors that regulate interactions between pancreatic tumors and their surrounding stroma are beginning to be identified. Yet broader physiological factors that influence these interactions remain unclear. Here, we discuss a series of preclinical and mechanistic studies that highlight the important role chronic stress plays as a physiological regulator of neural-tumor interactions in driving the progression of pancreatic cancer. These studies propose several approaches to target stress signaling via the β-adrenergic signaling pathway in order to slow pancreatic tumor growth and metastasis. They also provide evidence to support the use of β-blockers as a novel therapeutic intervention to complement current clinical strategies to improve cancer outcome in patients with pancreatic cancer.

Distinct patterns of peritumoral histological findings in subtypes of intraductal papillary mucinous neoplasms of the pancreas.

Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas are primary pancreatic neoplasms that can act as precursors to invasive adenocarcinoma of the pancreas. The peritumoral stroma has been increasingly recognized to play an important role in many types of tumors. Therefore, to investigate the clinicopathological significance of the peritumoral stroma in IPMNs, we examined the histological features of the peritumoral stroma in each subtype and histological grade of IPMNs. Eighty-two patients with IPMN, who underwent surgical resection, were reviewed clinicopathologically. Intraductal papillary mucinous neoplasms (86 lesions in total) were histologically subclassified into gastric (n = 51), intestinal (n = 22), pancreatobiliary (n = 11), and oncocytic (n = 2) subtypes. Peritumoral histological features between the gastric and intestinal subtypes were evaluated by each histological grade. The results showed that subepithelial edema and inflammatory cell infiltration were more commonly observed in the gastric subtype (74% and 79%, respectively) than in the intestinal subtype (12% and 25%, respectively) of low-grade IPMNs. On the other hand, mucus lakes were more commonly observed in the intestinal subtype (100%) than in the gastric subtype (0%) of high-grade IPMNs. In addition, pancreatobiliary subtype IPMNs tended to exhibit acute inflammation such as neutrophil predominance. This study showed that peritumoral histological features differed among subtypes of IPMNs from low-grade tumors. These differences suggest the possibility that each subtype of IPMNs has a distinct mechanism from an early stage of tumor progression, which is reflected in the properties of the peritumoral stroma.

Immunostimulatory early phenotype of tumor-associated macrophages does not predict tumor growth outcome in an HLA-DR mouse model of prostate cancer.

Tumor-associated macrophages (TAM) were shown to support the progression of many solid tumors. However, anti-tumor properties of TAM were also reported in several types of cancer. Here, we investigated the phenotype and functions of TAM in two transgenic mouse models of prostate cancer that display striking differences in tumor growth outcome. Mice expressing prostate-specific antigen (PSA) as a self-antigen specifically in prostate (PSAtg mice) rejected PSA-expressing transgenic adenocarcinoma of mouse prostate (TRAMP) tumors. However, the introduction of HLA-DRB1*1501 (DR2b) transgene presenting PSA-derived peptides in a MHC class II-restricted manner exacerbated the growth of TRAMP-PSA tumors in DR2bxPSA F 1 mice. Despite the difference in tumor growth outcome, tumors in both strains were equally and intensively infiltrated by macrophages on the first week after tumor challenge. TAM exhibited mixed M1/M2 polarization and simultaneously produced pro-inflammatory (TNFα, IL1β) and anti-inflammatory (IL10) cytokines. TAM from both mouse strains demonstrated antigen-presenting potential and pronounced immunostimulatory activity. Moreover, they equally induced apoptosis of tumor cells. In vivo depletion of macrophages in DR2bxPSA F 1 but not PSAtg mice aggravated tumor growth suggesting that macrophages more strongly contribute to anti-tumor immunity when specific presentation of PSA to CD4+ T cells is possible. In summary, we conclude that in the early stages of tumor progression, the phenotype and functional properties of TAM did not predict tumor growth outcome in two transgenic prostate cancer models. Furthermore, we demonstrated that during the initial stage of prostate cancer development, TAM have the potential to activate T cell immunity and mediate anti-tumor effects.

RARβ Promoter Methylation as an Epigenetic Mechanism of Gene Silencing in Non-small Cell Lung Cancer.

The retinoid acid receptor-p (RARβ) gene is one of the tumor suppressor genes (TSGs), which is frequently deleted or epigenetically silenced at an early stage of tumor progression. In this study we investigated the promoter methylation and expression status of the RARβ gene in 60 surgically resected non-small cell lung cancer (NSCLC) tissue samples and 60 corresponding unchanged lung tissue samples, using methylation-specific PCR and real-time-polymerase chain reaction (qPCR) techniques. We correlated the results with the pathological features of tumors and clinical characteristics of patients. qPCR analysis detected a significantly lower RARβ expression in the patients with adenocarcinoma (AC) and large cell carcinoma (LCC) than in those with squamous cell carcinoma (SCC) (AC vs. SCC, p = 0.032; AC and LCC vs. SCC, p = 0.0 13). Additionally, significantly lower expression of the RARβ gene was revealed in the patients with non-squamous cell cancer with a history of smoking assessed as pack-years (PY < 40 vs. PY ≥ 40, p = 0.045). Regarding RARβ promoter methylation, we found significant differences in the methylation index in the SCC group when considering pTNM staging; with higher index values in T1a + T1b compared with T2a + T2b and T3 + T4 groups (p = 0.024). There was no correlation between the methylation status and expression level of the RARβ gene, which suggests that other molecular mechanisms influence the RARβ expression in NSCLC patients. In conclusion, different expression of the RARβ gene in SCC and NSCC makes the RARβ gene a valuable diagnostic marker for differentiating the NSCLC subtypes.

Abstract 1668: Stabilin-1 is expressed on tumor-associated macrophages in breast cancer and supports tumor growth in animal model of breast adenocarcinoma by clearance of SPARC

Abstract Tumor-associated macrophages (TAM) are crucial participants in malignant progression. Acquisition of M2 (alternatively activated) phenotype by TAM during tumor progression enhances the immunosuppressive and tumor-supportive properties of TAM resulting in tumor invasion, metastasis and angiogenesis. Previously we have found that stabilin-1, a multifunctional scavenger/sorting receptor, is a marker of M2 macrophages that is expressed by TAM in several murine tumor models. However, its role in tumor progression was not defined. In order to identify the role of stabilin-1 in tumor progression, the model of mammary adenocarcinoma (TS/A) was established in BALB/c mice with stabilin-1 knockout. The growth of TS/A mammary adenocarcinoma in stabilin-1 knockout (ko) mice was suppressed by 36%. To identify the role of stabilin-1 in TAM biology and reveal functions of stabilin-1 that are important for tumor progression, isolation of high purity TAM from TS/A murine adenocarcinoma was established and optimized. Flow cytometry analysis revealed that adhesion/internalisation of extracellular SPARC was decreased in the isolated stabilin-1 ko TAM compared to wt TAM. Immunofluorescent/confocal microscopy showed that transport of SPARC into the endocytic pathway was significantly impaired in the stabilin-1 ko TAM. Since SPARC is known to inhibit the development of solid tumors including breast cancer we hypothesize that knockout of stabilin-1 in TAM induces accumulation of extracellular SPARC resulting in suppression of tumor growth. Analysis of two cohorts of female patients with breast carcinoma of different stages demonstrated that stabilin-1 is expressed on significant part of tumor associated macrophages. Three types of TAM were identified by co-staining with anti-stabilin-1 RS1 antibody and anti-CD68 antibody: CD68+stabilin-1-, CD68+stabilin-1+ and CD68-stabilin-1+. The highest levels of stabilin-1 expression and highest amount of stabilin-1+ TAM were found on stages I and IIa, suggesting that stabilin-1 is required for tumor growth at early stages of tumor progression. Our data indicate that stabilin-1 expression on TAM is necessary on the early stages of tumor growth in human cancer. Genetic knockout of stabilin-1 results in decrease in tumor growth in mouse adenocarcinoma model. In addition, tumor-associated macrophages deficient in stabilin-1 expression have significantly decreased ability for the endocytic clearance of SPARC. Citation Format: Vladimir Ryabov, Ilja Ovsij, Aida Avdic, Kai Schledzewski, Alexei Gratchev, Nan Wang, Bernd Arnold, Sergij Goerdt, Frederick Pfister, Alexander Marx, Limin Zheng, Julia Kzhyshkowska. Stabilin-1 is expressed on tumor-associated macrophages in breast cancer and supports tumor growth in animal model of breast adenocarcinoma by clearance of SPARC. [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 1668. doi:10.1158/1538-7445.AM2014-1668

Immunostimulatory early phenotype of tumor-associated macrophages does not predict tumor growth outcome in the HLA-DR mouse model of prostate cancer (TUM4P.923)

Abstract Tumor-associated macrophages (TAM) were shown to support progression of many solid tumors. However, anti-tumor properties of TAM were also reported in several types of cancer. Here, we investigated the phenotype and functions of TAM in two transgenic mouse models of prostate cancer displaying striking differences in the tumor growth outcome. Mice expressing Prostate-Specific Antigen as a self-antigen in the prostate (PSA tg mice) rejected PSA-expressing TRAMP tumors with high frequency. However, introduction of HLA-DRB1*1501(DR2b) transgene aggravated the growth of TRAMP-PSA tumors in DR2bxPSA F1 mice. Despite the difference in the tumor growth outcome, tumors in both strains were equally and intensively infiltrated by TAM at the first week after tumor challenge. TAM exhibited mixed M1/M2 polarization characterized by the production of pro-inflammatory (TNFα, IL1β) and anti-inflammatory (IL10) cytokines. They also secreted T cell/NK cell-recruiting chemokines (CXCL10, CCL2, CCL3, CCL4, CCL5) and expressed high levels of MHC class II and co-stimulatory molecules. Functionally, TAM demonstrated pronounced immunostimulatory activity in a mixed leukocyte reaction and induced apoptosis of tumor cells. We conclude that during early stages of tumor progression, the phenotype and functional properties of TAM did not predict tumor growth outcome in our model. Furthermore, we demonstrate that at this stage TAM have a potential to activate T cell immunity and mediate anti-tumor effects.

Long range epigenetic silencing is a trans-species mechanism that results in cancer specific deregulation by overriding the chromatin domains of normal cells

DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unknown whether there is a coordinated regulation of the genes embedded in these regions in normal cells and in early stages of tumor progression. To better characterize the molecular events associated with the regulation and remodeling of these regions we analyzed two regions undergoing LRES in human colon cancer in the mouse model. We demonstrate that LRES also occurs in murine cancer in vivo and mimics the molecular features of the human phenomenon, namely, downregulation of gene expression, acquisition of inactive histone marks, and DNA hypermethylation of specific CpG islands. The genes embedded in these regions showed a dynamic and autonomous regulation during mouse intestinal cell differentiation, indicating that, in the framework considered here, the coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign adenomas in ApcMin/+ mice showed overexpression of most of the genes affected by LRES in cancer, which suggests that the repressive remodeling of the region is a late event. Chromatin immunoprecipitation analysis of the transcriptional insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain boundaries as compared with normal cells. Malignant regression of cancer cells by in vitro differentiation resulted in partial reversion of LRES and gain of CTCF binding. We conclude that genes in LRES regions are plastically regulated in cell differentiation and hyperproliferation, but are constrained to a coordinated repression by abolishing boundaries and the autonomous regulation of chromatin domains in cancer cells.

Elevated Maspin Expression Is Associated with Better Overall Survival in Esophageal Squamous Cell Carcinoma (ESCC)

Tumor suppressor maspin is a differentially regulated gene in the progression of many types of cancer. While the biological function of maspin in blocking tumor invasion and metastasis is consistent with the loss of maspin expression at the late stage of tumor progression, the differential expression and the biological significance of maspin in early stage of tumor progression appear to be complex and remain to be elucidated. In the current study, we examined the expression of maspin in 84 esophageal squamous cell carcinoma (ESCC) cases (stages I–III) and 55 non-tumor adjacent esophageal tissue specimens by immunohistochemical (IHC) staining. The correlation of maspin with clinicopathological parameters was analyzed. Compared to normal esophageal squamous tissue where 80% (47/55) of the cases expressed maspin at a low to moderate level, all ESCC specimens (100% (84/84)) were positive for maspin expression at a moderate to high level. ESCC with low or moderate maspin expression had significantly shorter postoperative survival rates compared to those that had high maspin expression (p<0.001). Since the correlation of maspin with ESCC histology and the correlation of maspin with ESCC prognosis seem to be at odds, we further investigated the biological function of maspin in ESCC using the established ESCC cell lines. The expression of maspin in five human esophageal squamous cancer cell lines (T12, E450, KYSE150, EC109, and KYSE510) was examined by the Western blot. ESCC cell line KYSE510 that did not express maspin and was stably transfected by maspin cDNA or an empty vector. The resulting transfected cells were characterized in vitro. Maspin expression significantly inhibited cell proliferation, motility and matrigel invasion. Taken together, our data suggest that the transient up-regulation of maspin in the early development of ESCC may be a defense mechanism against further transition towards more malignant phenotypes, ultimately slowing down ESCC tumor progression.

Targeted Therapy; From Advanced Melanoma to the Adjuvant Setting

Targeted Therapy; From Advanced Melanoma to the Adjuvant Setting

MiR-203 Regulates Cell Proliferation through Its Influence on Hakai Expression

Gene expression is potently regulated through the action of microRNAs (miRNAs). Here, we present evidence of a miRNA regulating Hakai protein. Hakai was discovered as an E3 ubiquitin-ligase that mediates the posttranslational downregulation of E-cadherin, a major component of adherens junctions in epithelial cells and a potent tumour suppressor. Recent data have provided evidence that Hakai affects cell proliferation in an E-cadherin-independent manner, thus revealing a role for Hakai in the early stages of tumour progression. Furthermore, Hakai is highly up-regulated in human colon adenocarcinomas compared to normal tissues. However, the molecular mechanisms that regulate Hakai abundance are unknown. We identified two putative sites of miR-203 interaction on the Hakai mRNA, in its 3′-untranslated region (UTR). In several human carcinoma cell lines tested, overexpression of a miR-203 precursor (Pre-miR-203) reduced Hakai abundance, while inhibiting miR-203 by using an antisense RNA (Anti-miR-203) elevated Hakai levels. The repressive influence of miR-203 on the Hakai 3′-UTR was confirmed using heterologous reporter constructs. In keeping with Hakai's proliferative influence, Anti-miR-203 significantly increased cell number and BrdU incorporation, while Pre-miR-203 reduced these parameters. Importantly, the growth-promoting effects of anti-miR-203 required the presence of Hakai, because downregulation of Hakai by siRNA suppressed its proliferative action. Finally, in situ hybridization showed that miR-203 expression is attenuated in colon tumour tissues compared to normal colon tissues, suggesting that miR-203 could be a potential new prognostic marker and therapeutic target to explore in colon cancer. In conclusion, our findings reveal, for the first time, a post-transcriptional regulator of Hakai expression. Furthermore, by lowering Hakai abundance, miR-203 also reduces Hakai-regulated-cell division.

Progressive growth of a murine T cell lymphoma alters population kinetics and cell viability of macrophages in a tumor-bearing host

Tumor progression induces infiltration of immune cell populations at the site of tumor growth. Infiltrated leukocyte population including monocyte and macrophages interacts with tumor cells and tumor microenvironment and results in the suppression of macrophage functions. Impaired functions of macrophages result in the suppression/inhibition of cell-mediated immunity leading to inefficient antitumor immune responses. Impaired macrophage population invariably helps in immune selection of tumor leading to uninterrupted growth and progression in the host. Murine T cell lymphoma designated as Dalton's lymphoma is highly immunosuppressive and invasive tumor of T cell origin, which completely paralyzes the host's immune system resulting in a very short life span of the host. Progressive growth of Dalton's lymphoma (DL) cells has been known to inhibit the release of inflammatory cytokines and effector mediator molecules. In this study, we demonstrate that intraperitoneal transplant of DL cells in normal healthy host induces a rapid increase in macrophage cell population during early stage of tumor progression and progressive decrease in tumor-associated macrophage population and reduced survival of macrophages in advance stage of tumor burden.

Unexpected Dissemination Patterns in Lymphoma Progression Revealed by Serial Imaging within a Murine Lymph Node

Non-Hodgkin lymphoma (NHL) is a heterogeneous and highly disseminated disease, but the mechanisms of its growth and dissemination are not well understood. Using a mouse model of this disease, we used multimodal imaging, including intravital microscopy (IVM) combined with bioluminescence, as a powerful tool to better elucidate NHL progression. We injected enhanced green fluorescent protein and luciferase-expressing Eμ-Myc/Arf(-/-) (Cdkn2a(-/-)) mouse lymphoma cells (EL-Arf(-/-)) into C57BL/6NCrl mice intravenously. Long-term observation inside a peripheral lymph node was enabled by a novel lymph node internal window chamber technique that allows chronic, sequential lymph node imaging under in vivo physiologic conditions. Interestingly, during early stages of tumor progression we found that few if any lymphoma cells homed initially to the inguinal lymph node (ILN), despite clear evidence of lymphoma cells in the bone marrow and spleen. Unexpectedly, we detected a reproducible efflux of lymphoma cells from spleen and bone marrow, concomitant with a massive and synchronous influx of lymphoma cells into the ILN, several days after injection. We confirmed a coordinated efflux/influx of tumor cells by injecting EL-Arf(-/-) lymphoma cells directly into the spleen and observing a burst of lymphoma cells, validating that the burst originated in organs remote from the lymph nodes. Our findings argue that in NHL an efflux of tumor cells from one disease site to another, distant site in which they become established occurs in discrete bursts.

Abstract B28: Evidence for the antagonistic form of CXCL10 in high grade serous epithelial ovarian tumours

Abstract The chemokine CXCL10 is a potent angiostatic and chemoattractive agent, whose primary role is to recruit activated T-cells into sites of inflammation. We have investigated the expression of CXCL10 in serous epithelial ovarian cancers (SOC), with a focus on specific post-translational modifications that control its function. The study group was comprised of a small cohort of patients diagnosed with high grade (HGSOC) or low grade (LGSOC) ovarian tumors, benign SOC, non-ovarian benign gynecological disease or no pathology. Patients were post-menopausal and had not undergone any prior treatment. CXCL10 expression in tumor tissue was assessed by quantitative PCR and western blotting. Immunoassay was used to analyse plasma CA125, and plasma and urine CXCL10 levels in matched patient samples. Localization and correlation with leukocyte recruitment was determined using immunohiostochemical staining of tumor tissue for CXCL10 and the leukocyte cell surface antigen CD45. Proteolytic processing of CXCL10 was analyzed using MALDI imaging mass spectrometry (IMS), and de novo sequence information obtained by immunoprecipitation and MS/MS analysis. We identified significantly elevated expression of both CXCL10 mRNA and protein in HGSOC, with the highest levels evident in early stage (FIGO stage Ib-c) disease. CXCL10 expression was localized to the cytoplasm of tumor epithelial cells. Despite high levels of CXCL10, however, significant leukocyte infiltration was only observed in low grade tumors or within benign cysts; HGSOC contained significantly fewer CD45+ infiltrating cells in tumor tissue. The proteolytic removal of 2 N-terminal amino acids converts CXCL10 into a potent antagonist of chemotaxis and T-cell recruitment. We therefore investigated the presence of proteolytic modifications to CXCL10 using mass spectrometry. MALDI IMS revealed masses suggesting a 2-amino acid and a 9-amino acid truncation at the N- or C-termini of CXCL10, respectively. These masses also displayed strikingly different localization; N-terminally truncated CXCL10 was present only in tumor epithelium, whilst C-terminally truncated CXCL10 was located only in the intervening regions of stromal tissue. Immunoprecipitation and MS/MS analysis confirmed the presence of N-terminally truncated CXCL10 in HGSOC. Our data identify, for the first time, the presence of the “antagonistic” form of CXCL10 in HGSOC. Antagonistic CXCL10 is present at a clinically early stage of tumor progression and correlates with poor leukocyte infiltration into tumors, suggestive of a direct influence on patient outcomes. We hypothesize that tumor-specific conversion of CXCL10 from agonist to antagonist represents a novel mechanism by which HGSOC may partially evade the early immune response. Our data also suggest inhibition of CXCL10 cleavage may represent a new avenue for the treatment of patients diagnosed with HGSOCs. Citation Format: Andrew N. Stephens, Jyothsna R. Rao, Santanu Deb-Choudhury, Adam Rainczuk. Evidence for the antagonistic form of CXCL10 in high-grade serous epithelial ovarian tumors. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B28.

Abstract 819: Chronic smoking increases tumorigenicity and imparts resistance to platinum-based chemotherapy partly by down-regulating Smad3 in non-small cell lung cancer

Abstract Inactivating mutations in TGF-β receptors and Smad signal transducers that contribute to resistance to TGF-β, are associated with only very small number of NSCLC. The Smad3/Smad4 pathway is important for TGF-β-induced tumor suppressor functions like growth inhibition and apoptosis in normal epithelium and in the early stages of tumor progression. Epidemiological studies have demonstrated that most cases of lung cancers (85-90%) are directly attributable to cigarette smoking. However, nothing is known how smoking is involved in inhibiting tumor suppressor functions of TGF-ß and whether or how the TGF-β signaling proteins might be involved in the chemo resistance of platinum-based drugs in smokers. To address this issue, lung adenocarcinoma A549 and immortalized bronchial epithelial HPL1A cells were chronically treated (300 days) with cigarette smoke condensate (CSC) and dimethyl sulphoxide, as a control to mimic the conditions of long-term cigarette smoking. Prolonged exposure of these cells to CSC resulted in a decreased Smad-mediated TGF-β- signaling due to reduced expression of Smad3 through histone deacetylation. Long-term CSC treatment reduced apoptosis, increased cell viability; decreased TGF-β-mediated growth inhibition, and enhanced tumorigenicity both in vitro and in vivo. Re-expression of Smad3 in the long- term CSC treated cells reversed the cancerous phenotypes observed due to long- term CSC treatment. Immunohistochemical staining of tissue microarrays revealed that Smad3 expression is lower in smokers compared to non-smokers. This is supported by the results from public databases. However, low expression of Smad3 is not a prognostic biomarker to predict the survival outcome of patients with lung cancer. The long long-term CSC treatment also rendered the cells resistant to platinum-based chemotherapy by up-regulating Bcl2. Blocking the effect of Bcl2 both by small molecule inhibitor ABT-737 and siRNA approaches re-sensitized the cells to platinum-based chemotherapy. The re-expression of exogenous Smad3 in the long long-term CSC treated cells decreased the Bcl2 levels and re-sensitized the cells to platinum-based chemotherapy. Thus, down regulation of Smad3 by smoking conditions increases the expression of Bcl2 resulting in the resistance to platinum- based drugs in our model system. Collectively, these data provide evidence that cigarette smoking promotes tumorigenicity and makes the cells resistant to platinum-based chemotherapy by abrogating apoptosis, partly by reducing the expression of Smad3. In the current era of personalized medicine, there is a pressing need to identify predictive biomarkers for stratifying smokers to predict the response to contemporary platinum-based chemotherapy. The low expression of Smad3 can be a putative predictive biomarker of smokers for platinum-based chemotherapy. 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 819. doi:1538-7445.AM2012-819

Abstract SY04-02: Reaching escape velocity: Using lineage tracing to study EMT and dissemination in pancreatic cancer

Abstract Metastastic spread has been difficult to study because it involves a series of rare events that are difficult to observe in vivo. To capture such events, we have employed lineage tracing - a technique which allows cells to be tagged and tracked - and applied it to an autochthonous mouse model of pancreatic cancer. Using this approach, found that pancreas-derived cells entered the circulation at the PanIN-stage, prior to the presence of a histologically recognizable tumor. Moreover, invasive behavior was almost universally associated with epithelial-to-mesenchymal transition (EMT), and bloodstream entry was associated with a “cancer stem cell” phenotype. These observations demonstrate that cellular dissemination in pancreatic cancer can occur at a very early stage of tumor progression. 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 SY04-02. doi:1538-7445.AM2012-SY04-02

Telomerase activity, HPV presence and splice pattern of hTERT mRNA in cervical intraepithelial neoplasias and adjacent normal tissues

Cervical cancers are characterized by the persistence of human papilloma virus (HPV) genome that is found in tissue samples starting from the early stages of tumor progression. Just like in other tumors, the activation of telomerase was observed in cervical carcinomas, but information about its expression was controversial. On model systems (cell lines) alternate splicing of human telomerase reverse transcriptase mRNA may take part in telomerase activity regulation.The aim of this study was to find possible correlations between the presence of HPV sequences, activity of telomerase and expression of different spliced forms of hTERT RNA in cervical intraepithelial neoplasias (CIN). The results show that HPV DNA is present in 60% of normal tissue adjacent to CIN lesions and up to 84% in CIN samples. Telomerase activity was found in 28% of adjacent normal tissue and in 68% of CIN II–III. hTERT RNA that encodes an active enzyme was present almost in all CIN samples. Variations in levels of telomerase activity are presumably not regulated by the splicing forms of hTERT mRNA with deletions.

TRAF6 Mediates IL-1β/LPS-Induced Suppression of TGF-β Signaling through Its Interaction with the Type III TGF-β Receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-βsignaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.

A vascular niche and a VEGF–Nrp1 loop regulate the initiation and stemness of skin tumours

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF's ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.