Mediate Tumor Progression Research Articles

Abstract 1557: Tumor Galectin-1 mediates lung cancer growth and metastasis through immunoregulation of T-cell apoptosis

Abstract Studies have shown that Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced under hypoxia, can promote tumor aggressiveness through its effect on angiogenesis and T-cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is unclear. Using matched Gal-1 wild-type and null mice together with Lewis lung carcinoma cells stably knocked-down of Gal-1 (shGal-1) or scramble control (Scr), we show that tumor Gal-1 is more critical that host Gal-1 in promoting tumor growth and spontaneous metastasis. Scr tumors had higher growth and metastasis rates compared to shGal-1 tumors, regardless of the host genotype. The enhanced growth and metastasis associated with Gal-1 appears to be related to its immunomodulatory function since it was abolished in immune-deficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between shGal-1 and Scr tumors when they were allowed to reach comparable size. These data indicated that tumor Gal-1 is more important than host Gal-1 in mediating tumor progression and that its effect was exerted through intratumoral T-cell modulation. These findings have broad implications in developing novel targeting strategies for Gal-1 in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1557. doi:10.1158/1538-7445.AM2011-1557

Abstract 1634: Targeting L1 cell adhesion molecule using lentivirus-mediated short hairpin RNA interference reverses aggressiveness of oral squamous cell carcinoma

Abstract Oral cancer consistently ranks as one of the ten most frequently diagnosed cancers in the world. Despite multidisciplinary treatment with surgery, chemotherapy, and radiation, more than 50% of patients with oral squamous cell carcinoma (OSCC) receiving therapy will eventually develop recurrent and metastatic disease, which carries a particularly poor prognosis. Characterization of the specific molecular alternations associated with OSCC cell growth, invasion, and metastasis can advance our understanding of the molecular mechanisms underlying cancer induction and progression as well provide a basis for the development of new and effective therapeutic treatments for cancer patients. The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in OSCC has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirusmediated small hairpin RNA (shRNA) led to a significant reduction in cell proliferation in vitro via retardation of cell cycle at the G1 phase. In addition, shRNA knockdown of L1CAM strongly attenuated the migration and invasion of SCC4 cells, and this was also observed to parallel increased E-cadherin levels and decreased levels vimentin, fibronectin, and Snail-family transcription factors, indicating that L1CAM expression was related to the epithelial-mesenchymal transition. Furthermore, while mice receiving orthotopically placed control SCC4 cells died within 40 days due to invasive tumor growth and regional lymph node metastasis, prolonged animal survival and complete suppression of tumor progression was observed in mice implanted with L1CAM-deficent SCC4 cells, further substantiating the fundamental importance of L1CAM in OSCC pathophysiology. Our findings suggested that L1CAM is a critical mediator of tumor progression in OSCC, and targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2011-1634

Suppression of dual-specificity phosphatase–2 by hypoxia increases chemoresistance and malignancy in human cancer cells

Hypoxia inducible factor-1 (HIF-1) is the master transcriptional regulator of the cellular response to altered oxygen levels. HIF-1α protein is elevated in most solid tumors and contributes to poor disease outcome by promoting tumor progression, metastasis, and resistance to chemotherapy. To date, the relationship between HIF-1 and these processes, particularly chemoresistance, has remained largely unexplored. Here, we show that expression of the MAPK-specific phosphatase dual-specificity phosphatase-2 (DUSP2) is markedly reduced or completely absent in many human cancers and that its level of expression inversely correlates with that of HIF-1α and with cancer malignancy. Analysis of human cancer cell lines indicated that HIF-1α inhibited DUSP2 transcription, which resulted in prolonged phosphorylation of ERK and, hence, increased chemoresistance. Knockdown of DUSP2 increased drug resistance under normoxia, while forced expression of DUSP2 abolished hypoxia-induced chemoresistance. Further, reexpression of DUSP2 during cancer progression caused tumor regression and markedly increased drug sensitivity in mice xenografted with human tumor cell lines. Furthermore, a variety of genes involved in drug response, angiogenesis, cell survival, and apoptosis were found to be downregulated by DUSP2. Our results demonstrate that DUSP2 is a key downstream regulator of HIF-1-mediated tumor progression and chemoresistance. DUSP2 therefore may represent a novel drug target of particular relevance in tumors resistant to conventional chemotherapy.

The HIF-1–Inducible Lysyl Oxidase Activates HIF-1 via the Akt Pathway in a Positive Regulation Loop and Synergizes with HIF-1 in Promoting Tumor Cell Growth

Adaptation to hypoxia is a driving force for tumor progression that leads to therapy resistance and poor clinical outcome. Hypoxic responses are mainly mediated by hypoxia-inducible transcription factor-1 (HIF-1). One critical HIF-1 target mediating tumor progression is lysyl oxidase (LOX), which catalyzes cross-linking of collagens and elastin in the extracellular matrix, thereby regulating tissue tensile strength. Paradoxically, LOX has been reported to be both upregulated and downregulated in cancer cells, especially in colorectal cancer. Thus, we hypothesized that LOX might regulate expression of HIF-1 to create a self-timing regulatory circuit. Using human colorectal carcinoma cell lines in which HIF-1 and LOX expression could be modulated, we showed that LOX induction enhanced HIF-1 expression, whereas LOX silencing reduced it. Mechanistic investigations revealed that LOX activated the PI3K (phosphoinositide 3-kinase)-Akt signaling pathway, thereby upregulating HIF-1α protein synthesis in a manner requiring LOX-mediated hydrogen peroxide production. Consistent with these results, cancer cell proliferation was stimulated by secreted and active LOX in an HIF-1α-dependent fashion. Furthermore, nude mice xenograft assays established that HIF-1 potentiated LOX action on tumor growth in vivo. Taken together, these findings provide compelling evidence that LOX and HIF-1 act in synergy to foster tumor formation, and they suggest that HIF-1/LOX mutual regulation is a pivotal mechanism in the adaptation of tumor cells to hypoxia.

Regulation of Nm23-H1 and Cell Invasiveness by Kaposi's Sarcoma-Associated Herpesvirus

The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and the induction of an invasive cellular phenotype by KSHV following de novo infection is an important pathogenic component mediating tumor progression. The metastasis suppressor gene known as Nm23-H1 regulates tumor cell invasiveness, but whether KSHV itself regulates Nm23-H1 expression or subcellular localization, and whether this impacts cell invasiveness, has not been established. We found that KSHV increases expression and nuclear translocation of Nm23-H1 and that nuclear translocation of Nm23-H1 is regulated by the KSHV-encoded latency-associated nuclear antigen (LANA). Moreover, activation of the Ras-BRaf-MAPK (mitogen-activated protein kinase) signal transduction pathway, secretion of promigratory factors associated with this pathway, and cell invasiveness are dependent on KSHV regulation of Nm23-H1. Finally, induction of cytoplasmic overexpression of Nm23-H1 using a pharmacologic inhibitor of DNA methylation reduced KSHV-associated Ras-BRaf-MAPK pathway activation and suppressed KSHV-induced invasiveness. These data provide the first evidence for KSHV regulation of Nm23-H1 as a mechanism for KSHV induction of an invasive cellular phenotype and support the potential utility of targeting Nm23-H1 as a therapeutic approach for the treatment of KS.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

C-Jun N-terminal Kinase 2 Regulates Multiple Receptor Tyrosine Kinase Pathways in Mouse Mammary Tumor Growth and Metastasis

c-Jun N-terminal kinase 2 (JNK2) isoforms are transcribed from the jnk2 gene and are highly homologous with jnk1 and jnk3 transcriptional products. JNK proteins mediate cell proliferation, stress response, and migration when activated by a variety of stimuli, including receptor tyrosine kinases (RTKs), but their ability to influence tumor metastasis is ill defined. To evaluate JNK2 in this manner, we used the highly metastatic 4T1.2 mammary tumor cells. Short hairpin RNA expression directed toward JNK2 (shJNK2) decreases tumor cell invasion. In vivo, shJNK2 expression slows tumor growth and inhibits lung metastasis. Subsequent analysis of tumors showed that shJNK2 tumors express lower GRB2-associated binding protein 2 (GAB2). In vitro, knockdown of JNK2 or GAB2 inhibits Akt activation by hepatocyte growth factor (HGF), insulin, and heregulin-1, while phosphorylation of ERK is constitutive and Src dependent. Knockdown of GAB2 phenocopies knockdown of JNK2 in vivo by reducing tumor growth and metastasis, supporting that JNK2 mediates tumor progression by regulating GAB2. The influence of jnk2 in the host or microenvironment was also evaluated using syngeneic jnk2-/- and jnk2+/+ mice. Jnk2-/- mice experience longer survival and less bone and lung metastasis compared to jnk2+/+ mice after intracardiac injection of 4T1.2 cells. GAB2 has previously been shown to mediate osteoclast differentiation, and osteoclasts are critical mediators of tumor-related osteolysis. Thus, studies focusing on the role of JNK2 on osteoclast differentiation were undertaken. ShJNK2 expression impairs osteoclast differentiation, independently of GAB2. Further, shJNK2 4T1.2 cells express less RANKL, a stimulant of osteoclast differentiation. Together, our data support that JNK2 conveys Src/phosphotidylinositol 3-kinase (PI3K) signals important for tumor growth and metastasis by enhancing GAB2 expression. In osteoclast progenitor cells, JNK2 promotes differentiation, which may contribute to the progression of bone metastasis. These studies identify JNK2 as a tumor and host target to inhibit breast cancer growth and metastasis.

Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression

Molecular-genetic imaging is advancing from a valuable preclinical tool to guiding patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression, protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 promoter (PEG-Prom), derived from a rodent gene mediating the malignant phenotype, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in murine models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter, tumor specificity and capacity for clinical translation, PEG-Prom-driven gene expression may represent a practical, new system for facilitating cancer imaging and therapy.

Abstract CN10-03: Stromal signals from the involuting microenvironment that are important in postpartum breast cancer

Abstract Prognosis of young women's breast cancer is influenced by pregnancy. Women diagnosed within 5 years of childbirth have worse prognosis than age-matched nulliparous women or women diagnosed while pregnant. These data indicate that pregnancy-associated breast cancer (PABC) exists as two subtypes, with poor prognosis associated with cases diagnosed in the postpartum setting rather than during pregnancy. We propose that breast involution following pregnancy accounts for the poor prognosis of breast cancers diagnosed within 5 years of childbirth. Characterization of the involuting mammary gland has identified tissue remodeling programs associated with wound healing and inflammatory responses that share similarities with cancer microenvironments known to be tumor promotional. Based on these results, we proposed the Involution Hypothesis, which states that the physiologically normal microenvironment of the postpartum involuting gland is tumor promotional. We estimate a high proportion of young women with breast cancer actually have a “recent” pregnancy as a negative prognostic feature. Specifically, with an upper limit of 40 years of age for reproductive potential and a recent pregnancy defined as within 5 years of diagnosis, an estimated 20,000 cases of breast cancer per year in the U.S. may qualify as postpartum. Targeting the postpartum window with interventions designed to reduce the tumor promotional attributes of involution is highly attractive, as this window of risk is restricted in time and the population is readily identifiable. Testing the Involution Hypothesis has been limited by lack of preclinical models. We describe here a mouse xenograft model of PABC that isolates postpartum mammary gland involution as a driving force for breast cancer progression and metastasis. In this model, human mammary MCFDCIS.com tumor cells injected into the postpartum, involuting mammary gland form more proliferative and invasive tumors than tumor cells injected into the nulliparous gland. The involuting gland is characterized by high levels of radiating collagen fibers organized similar to collagen associated with invasive tumors across species. In our model of postpartum breast cancer, fibrillar collagen is identified as a primary mediator of tumor progression. Mammary tumors arising in the collagen-rich, involuting microenvironment are invasive and have increased levels of COX-2. In vitro, primary tumor cells isolated from PABC tumors are motile and invasive in a COX-2 dependent manner. Evidence that COX-2 induced motility is dependent on collagen signaling is demonstrated with functional blocking antibodies against β1-integrin, 2β1-integrin, and integrin associated protein CD47. These data suggest that treatment targeting COX-2 during postpartum involution may decrease progression of postpartum breast cancer. To test this hypothesis, mice were treated with the NSAID ibuprofen for two weeks coinciding with postpartum mammary gland involution. NSAID treatment was found to reduce tumor size, COX-2 expression, and lung cell seeding in this model. These antitumor effects were observed without blocking apoptosis of secretory mammary epithelial cells, which is required to remodel the gland to a nonsecretory state, and is a requisite for advancing this strategy to clinical trial. The question of whether an NSAID based intervention study could be aimed at recently pregnant women at high risk for breast cancer remains to be determined, but is an extremely desirable objective given that there are more than 6 million pregnancies in the U.S. per year. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN10-03.

Hyaluronic Acid/Chitosan-g-Poly(ethylene glycol) Nanoparticles for Gene Therapy: An Application for pDNA and siRNA Delivery

To design hyaluronic acid (HA) and chitosan-g-poly(ethylene glycol) (CS-g-PEG) nanoparticles intended for a broad range of gene delivery applications. Nanoparticles formulated at different HA/CS-g-PEG mass ratios were developed to associate either pDNA or siRNA. The physico-chemical characteristics, morphology, association efficiency and nuclease protection ability of the nanocarriers were compared for these two molecules. Their biological performance, including transfection effciency, nanoparticle cellular uptake and citotoxicity, was assesed. The resulting nanoparticles showed an adequate size (between 130 and 180nm), and their surface charge could be modulated according to the nanoparticle composition (from +30mV to -20mV). All prototypes exhibited a greater association efficiency and nuclease protection for pDNA than for siRNA. However, cell culture experiments evidenced that HA/CS-g-PEG nanoparticles were effective carriers for the delivery of both, siRNA and pDNA, eliciting a biological response with minimal cytotoxicity. Moreover, experiments performed in the HEK-EGFP-Snail1 cell line showed the potential of the HA/CS-g-PEG nanoparticles to silence the expression of the Snail1 transcription factor, an important mediator in tumor progression. HA/CS-g-PEG nanoparticles can be easily modulated for the delivery of different types of gene molecules, offering great potential for gene therapy applications, as evidenced by their biological performance.

Glutamate Receptor GRIA3—Target of CUX1 and Mediator of Tumor Progression in Pancreatic Cancer

Previously, we identified the transcription factor CUX1 as an important modulator of invasion and resistance to apoptosis. Expression profiles suggested that CUX1 regulates a complex transcriptional program mediating tumor progression. We aimed to identify functionally relevant targets of CUX1 by using RNA interference (RNAi)-based loss-of-function screens. Therefore, we generated an RNAi library containing putative transcriptional targets of CUX1 identified by microarrays and performed cell viability screens. Using this approach, several CUX1 targets with effect on tumor cell viability were identified, including the glutamate receptor GRIA3, which was validated in detail for its effects on proliferation, apoptosis, and cell migration using RNAi knock-down and overexpression strategies in vitro, as well as xenograft models in vivo. The expression of GRIA3 was evaluated in human pancreatic cancer tissues. We found that knock-down of GRIA3 significantly reduced proliferation and migration and enhanced apoptosis. In contrast, overexpression of GRIA3 significantly reduced apoptosis and enhanced both proliferation and tumor cell migration. GRIA3 could be confirmed as a downstream effector of CUX1 and was expressed in pancreatic cancer tissues. In vivo, GRIA3 significantly enhanced the growth of subcutaneous xenografts. Inhibitors of glutamate receptors such as GYKI52466 and SYM2206 significantly decreased survival of pancreatic cancer cells, suggesting the presence of glutamate signaling in pancreatic cancer. In conclusion, GRIA3 plays a role as a mediator of tumor progression in pancreatic cancer downstream CUX1. To our knowledge, this is the first report to identify a glutamate receptor as a modulator of tumor progression in a solid cancer outside the brain.

Mutations in TGFbeta-RII and BAXmediate tumor progression in the later stages of colorectal cancer with microsatellite instability

BackgroundMicrosatellite instability (MSI) occurs in 15% of colorectal cancers (CRC). The genetic targets for mutation in the MSI phenotype include somatic mutations in the transforming growth factor beta receptor typeII (TGFbetaRII), BAX, hMSH3 and hMSH6. It is not clear how mutations of these genes mediate tumor progression in the MSI pathway, and the temporal sequence of these mutations remains uncertain. In this study, early stage CRCs were examined for frameshift mutations in these target genes, and compared with late stage tumors and CRC cell lines.MethodsWe investigated 6 CRC cell lines and 71 sporadic CRCs, including 61 early stage cancers and 10 late stage cancers. Mutations of repetitive mononucleotide tracts in the coding regions of TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR and Fas antigen were identified by direct sequencing.ResultsThirteen (18.3%) of 71 CRC, including 9/61 (14.7%) early stage cancers and 4/10 (40%) late stage cancers, were identified as MSI and analyzed for frameshift mutations. No mutation in the target genes was observed in any of the 9 early stage MSI CRCs. In contrast, frameshift mutations of TGFbetaRII, BAX, hMSH3 and hMSH6 were present in 3/4 late stage MSI tumors. There is a statistical association (p = 0.014) between mutation in any one gene and tumor stage.ConclusionsTGFbetaRII, BAX, hMSH3 and hMSH6 mutations are relatively late events in the genesis of MSI CRCs. The frameshift mutations in these target genes might mediate progression from early to late stage cancer, rather than mediating the adenoma to carcinoma transition.

Abstract 5320: Deletion of TGFß signaling in Gr-1+CD11b+ myeloid cells attenuates breast adenocarcinoma progression

Abstract TGFß is a very important mediator in tumor progression. However, the contribution of TGFß signaling in tumor-infiltrating inflammatory cells in this process is unclear. Gr-1+CD11b+ myeloid cells are overproduced in tumor host. They infiltrate into tumor tissues and contribute to immune suppression, tumor angiogenesis and metastasis. Data from our studies demonstrate that Gr-1+CD11b+ myeloid cells are recruited into the invasive front of breast adenocarcinomas. Tumor-infiltrating Gr-1+CD11b+ cells exhibit a significantly elevated TGFß signaling, including increased expression of TGFß type I and type II receptor, TGFß1 and TGFß3 ligands, as well as latent TGFß binding protein. They also produce a high level of multiple matrix metalloproteinases (MMPs) such as MMP13, MMP14, MMP2 and MMP9. Interestingly, genetic deletion of the gene encoding TGFß type II receptor (Tgfbr2) in Gr-1+CD11b+ cells significantly attenuates the production of MMP14 and MMP2. These in vitro studies were conducted by transduction of Gr-1+CD11b+ cells from Tgfbr2flox/flox mice (loxP-flanked Tgfbr2) with adenovirus expressing DNA recombinase. To further investigate if TGFß signaling plays an important role in Gr-1+CD11b+ myeloid cells mediated breast tumor progression, mice with conditional knockout of Tgfbr2 in Gr-1+CD11b+ cells are generated through cross breeding of Tgfbr2flox/flox mice with LysM-Cre mice expressing Cre in myeloid cells. These mutant mice are viable and appear to be normal. Southern blotting showed that Tgfbr2 deletion is restricted to bone marrow, spleen and lymph node but not other organs. Sorted Gr-1+CD11b+ myeloid cells but not T or B lymphoid cells exhibited over 85% of Tgfbr2 deletion. This specific TGF-ß inactivation in Gr-1+CD11b+ cells allows us to investigate its effect on tumor growth and angiogenesis, as well as metastasis. We observed a significantly attenuated 4T1 tumor growth in these knockout mice compared with the control littermates. We postulate that TGFß signaling in host derived Gr-1+CD11b+ cells is an important part of TGFß effect in tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5320.

Abstract 2059: The role of microRNA-196b in cervical cancer

Abstract Introduction: MicroRNAs (miRs) are ∼22-nucleotide, non-coding RNAs that regulate gene expression post-transcriptionally. Recent data have indicated that miRs are aberrantly expressed in almost all human malignancies, including cervical cancer. In order to better understand how miRs might mediate tumor progression in cervical cancer, dysregulated miRs were identified and functional studies performed for miR-196b. Experimental Procedures: miR expression was measured in 3 cervical cancer cell lines (SiHa, ME-180 and HT3) and 3 normal cervix tissues using a quantitative real-time PCR approach examining 365 miRs and 3 endogenous controls. SiHa cells were transfected with Pre-miR miRNA Precursor Molecules (Ambion) to determine the effects of miR-196b overexpression. Candidate miR-196b targets were elucidated using a tri-pronged approach combining in silico target prediction algorithms, clinical mRNA expression data, and in vitro mRNA expression data following transfection of cell lines. To test for direct binding of miR-196b to the HOXB7 3’ UTR, a luciferase reporter assay was performed. Reporter vectors were constructed containing either the HOXB7 3’ UTR sequence (pMIR-REPORT-HOXB7/UTR), or the HOXB7 3’ UTR sequence with a mutation in the predicted miR-196b binding site (pMIR-REPORT-HOXB7/mut). SiHa cells were serially transfected with: a) miR-196b pre-miR or negative control pre-miR, and b) pMIR-HOXB7/UTR or pMIR-HOXB7/mut, and luciferase activity was measured 24 hours later. To determine the effect of knocking down HOXB7, SiHa cells were transfected with siHOXB7 (Qiagen). Results: Twenty-seven miRs were observed to be downregulated in all three cell lines compared to normal cervix. One of the 27 downregulated miRs, miR-196b, was shown to inhibit cell growth and colony-forming ability when overexpressed in SiHa cells. HOXB7 was identified as one of the candidate targets of miR-196b using our tri-pronged approach. Knocking down HOXB7 recapitulated the inhibition in cell growth and clonogenicity that was observed following mir-196b overexpression. When the luciferase reporter assay was performed, miR-196b overexpression resulted in a significant reduction in luciferase activity in cells transfected with pMIR-HOXB7/UTR, but not in cells transfected with pMIR-HOXB7/mut, demonstrating direct and specific binding of miR-196b to the 3’ UTR of HOXB7. Conclusion: miR-196b is downregulated in cervical cancer and negatively regulates HOXB7. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2059.

Abstract 234: Transcription factor LSF is a novel oncogene for hepatocellular carcinoma (HCC)

Abstract While the incidence of overall cancers is decreasing, Hepatocellular Carcinoma (HCC) is one of the few cancers the incidence of which is on an increase. The mortality rate of HCC parallels that of incidence since HCC is a highly aggressive cancer with extremely limited currently available therapeutic strategies. Understanding the molecular mechanism of hepatocarcinogenesis will help develop novel, targeted and more effective therapeutic regimen to counteract this fatal disease. Here, we demonstrate that the cellular transcriptional factor Late SV40 Factor (LSF) functions as an oncogene in HCC. The level of LSF expression is significantly increased in human HCC patients compared to matched non-neoplastic controls and normal liver and LSF expression significantly correlates with the stages and grades of HCC. Ectopic overexpression of LSF in nontumorigenic human HCC cells leads to highly aggressive, multi-organ metastatic tumors while inhibition of LSF in aggressive human HCC cells profoundly inhibits tumorigenic and metastatic properties in athymic nude mice. Affymetrix microarray studies identified novel LSF-downstream genes that are associated with cell growth, angiogenesis, invasion, metastasis, chemoresistance, and senescence. Osteopontin, a key molecule mediating tumor progression, especially metastasis, was identified as a downstream target gene of LSF. Promoter-reporter and chromatin immunoprecipitation analyses confirmed that LSF transcriptionally upregulates OPN by binding to its promoter. Knockdown of OPN gene in LSF-overexpressing HCC cells markedly inhibits LSF-mediated tumor progression including metastasis. This is the first report on the novel function of LSF as an oncogene in any cancer indication. Since LSF expression correlates with the advanced stages of the disease, it might be employed as a diagnostic/prognostic marker for HCC. The DNA binding domain of LSF might be targeted with small molecule inhibitors that might be developed as potential HCC therapeutic. Our findings thus have significant translational implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 234.

Clinical significance of syndecan-1 and versican expression in human epithelial ovarian cancer

Proteoglycans are ubiquitous components of the extracellular matrix and cell surface, and may mediate tumor progression and metastasis. The aim of this study was to evaluate the expression of syndecan-1 and versican in epithelial ovarian cancer. We immunohistochemically evaluated the expression of syndecan-1 and versican in 111 patients with epithelial ovarian cancer, and analyzed the correlation of this expression with various observed clinicopathological features, including patient outcome. There is a significant correlation between primary and metastatic sites with respect to syndecan-1 and versican expression. Epithelial syndecan-1 expression was significantly lower in patients with advanced disease. Epithelial versican expression was significantly higher in patients with early disease, especially in clear cell adenocarcinoma patients. Stromal syndecan-1 and versican expression was significantly higher in patients with advanced disease. Multivariate analysis showed that negative epithelial syndecan-1 expression was an independent prognostic factor for progression-free survival. Stromal syndecan-1 and versican co-expression was of borderline significance for progression-free and overall survival. Loss of epithelial syndecan-1 expression and induction of stromal syndecan-1 and versican expression may be associated with tumor progression in epithelial ovarian cancer. Syndecan-1 and versican expression status can serve as an indicator of prognosis in patients with epithelial ovarian cancer.

Targeting of VEGF-dependent transendothelial migration of cancer cells by bevacizumab

Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression. We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF 165 as a mediator of effusion formation. VEGF 165 was constantly secreted by all cultured tumor cells in an mTOR-dependent manner, as it was inhibited by the mTOR inhibitor rapamycin. Secreted VEGF 165 showed functional activity by inducing endothelial leakiness and tumor cell-transendothelial migration in vitro, effects which could be reverted by the anti-VEGF antibody bevacizumab. Thus, mTOR inhibitors as well as bevacizumab should be considered as potential agents in cancer patients suffering from malignant effusions.

HMGA2: A Potential Biomarker Complement to P53 for Detection of Early-stage High-grade Papillary Serous Carcinoma in Fallopian Tubes

Before high-grade papillary serous carcinoma (HG-PSC) becomes invasive, it is believed to be a poorly defined short-lived precursor lesion. A recent characterization of serous tubal intraepithelial carcinoma (STIC) and of the p53 signature suggested that HG-PSC may follow a stepwise progression on cellular and molecular levels. High-mobility group AT-hook 2 (HMGA2), an oncofetal protein, is overexpressed in ovarian cancer. To test whether HMGA2 can be another valuable marker for STIC, we examined HMGA2 expression in 3 groups of patients: (1) 24 patients with STIC and its invasive counterpart, HG-PSC of the fallopian tubes, (2) 24 patients with HG-PSC of the ovaries but without STIC (positive control), and (3) 30 patients with cancer and normal fallopian tubes (negative control). We found that HMGA2 was overexpressed in 75% of patients with STIC, was coexpressed with p53 in more than 50% of patients, and was completely negative in the secretory cells of the 30 patients with normal fallopian tubes. Among 7 patients with cells negative for p53 staining, HMGA2 was positive in 5; among 6 patients whose tumor cells were negative for HMGA2 in STIC, 3 were positive for HMGA2 in the invasive component; about 70% of invasive HG-PSC tumor cells were immunoreactive for both HMGA2 and TP53. In invasive carcinoma, HMGA2 overexpression was correlated with p53 (r=0.45), indicating the role of HMGA2 in p53 mediated tumor progression. Our findings of immunoreactivity for HMGA2 may lead to a novel, useful biomarker to complement p53 in the detection of early-stage serous carcinoma.

Insulin-Sensitizing Therapy Attenuates Type 2 Diabetes–Mediated Mammary Tumor Progression

OBJECTIVEType 2 diabetes increases breast cancer risk and mortality, and hyperinsulinemia has been identified as a major factor linking these two diseases. Thus, we hypothesized that pharmacological reduction of elevated insulin levels would attenuate type 2 diabetes–mediated mammary tumor progression.RESEARCH DESIGN AND METHODSWe studied mammary tumor development in MKR+/+ mice, a nonobese, hyperinsulinemic mouse model of type 2 diabetes. MKR+/+ mice were either crossed with mice expressing the polyoma virus middle T oncogene specifically in the mammary gland or inoculated orthotopically with the mouse mammary tumor cell lines Met-1 and MCNeuA. MKR+/+ or control mice harboring tumors were treated with CL-316243, a specific β3-adrenergic receptor agonist, which sensitizes insulin action but has no direct effect on the mouse mammary epithelium or Met-1 and MCNeuA cells.RESULTSCL-316243 treatment significantly reduced the elevated insulin levels in MKR+/+ mice and, as a consequence, attenuated mammary tumor progression in the three tumor models tested. This effect was accompanied by reductions in phosphorylation of insulin and IGF-I receptors in transformed mammary tissue.CONCLUSIONSInsulin-sensitizing treatment is sufficient to abrogate type 2 diabetes–mediated mammary tumor progression. Therefore, early administration of insulin-sensitizing therapy may reduce breast cancer risk and mortality in patients with type 2 diabetes.

Abstract A40: Six1 is dependent on its coactivator Eya2 to mediate tumor progression in the MCF7 mammary carcinoma cell line

Abstract The homeobox transcription factor Six1 is a critical mediator of embryonic development. In adults, upregulation of Six1 has been associated with a number of cancers where it not only promotes proliferation and survival, contributing to tumorigenesis, but also upregulates the TGFβ pathway, brings about an EMT-like transformation, and promotes metastasis. The Eya coactivator family is critical for Six1-mediated transcriptional activation in development. Our research is aimed at identifying what role the Eya family plays in Six1-mediated breast cancer progression. Examination of publicly available microarray data sets revealed that although high levels of Six1 significantly correlates with a shortened time to metastasis in breast cancer patients, presence of the Eya1-Six1 or Eya2-Six1 transcriptional complex correlates with a further reduced time to metastasis as well as with a significant decrease in survival time in breast cancer patients. Utilizing Six1 overexpressing MCF7 breast carcinoma cells with stable knock down of Eya2, we have found that lack of Eya2 reverses Six1-induced upregulation of the TGFβ pathway. Additionally, most Six1-induced EMT properties, including increased β-catenin responsive transcription and increased levels of the mesenchymal marker Fibronectin, were reversed with loss of Eya2. Overall, these data implicate Eya2 as a critical coactivator that is necessary for most of the metastasis-associated properties induced by Six1. Thus, as Six1 and Eya are not normally expressed in most adult tissues, the Six1-Eya interaction may be a valuable future drug target whose inhibition should result in limited side effects while potently targeting the tumor and its associated metastases. Citation Information: Cancer Res 2009;69(23 Suppl):A40.