Uterine Cancer Cells Research Articles

Biologic Effects of Platelet-Derived Growth Factor Receptor α Blockade in Uterine Cancer

Platelet-derived growth factor receptor α (PDGFRα) expression is frequently observed in many kinds of cancer and is a candidate for therapeutic targeting. This preclinical study evaluated the biologic significance of PDGFRα and PDGFRα blockade (using a fully humanized monoclonal antibody, 3G3) in uterine cancer. Expression of PDGFRα was examined in uterine cancer clinical samples and cell lines, and biologic effects of PDGFRα inhibition were evaluated using in vitro (cell viability, apoptosis, and invasion) and in vivo (orthotopic) models of uterine cancer. PDGFRα was highly expressed and activated in uterine cancer samples and cell lines. Treatment with 3G3 resulted in substantial inhibition of PDGFRα phosphorylation and of downstream signaling molecules AKT and mitogen-activated protein kinase (MAPK). Cell viability and invasive potential of uterine cancer cells were also inhibited by 3G3 treatment. In orthotopic mouse models of uterine cancer, 3G3 monotherapy had significant antitumor effects in the PDGFRα-positive models (Hec-1A, Ishikawa, Spec-2) but not in the PDGFRα-negative model (OVCA432). Greater therapeutic effects were observed for 3G3 in combination with chemotherapy than for either drug alone in the PDGFRα-positive models. The antitumor effects of therapy were related to increased apoptosis and decreased proliferation and angiogenesis. These findings identify PDGFRα as an attractive target for therapeutic development in uterine cancer.

Cross-talk between EphA2 and BRaf/CRaf Is a Key Determinant of Response to Dasatinib

EphA2 is an attractive therapeutic target because of its diverse roles in cancer growth and progression. Dasatinib is a multikinase inhibitor that targets EphA2 and other kinases. However, reliable predictive markers and a better understanding of the mechanisms of response to this agent are needed. The effects of dasatinib on human uterine cancer cell lines were examined using a series of in vitro experiments, including MTT, Western blot analysis, and plasmid transfection. In vivo, an orthotopic mouse model of uterine cancer was utilized to identify the biologic effects of dasatinib. Molecular markers for response prediction and the mechanisms relevant to response to dasatinib were identified by using reverse phase protein array (RPPA), immunoprecipitation, and double immunofluorescence staining. We show that high levels of CAV-1, EphA2 phosphorylation at S897, and the status of PTEN are key determinants of dasatinib response in uterine carcinoma. A set of markers essential for dasatinib response was also identified and includes CRaf, pCRaf(S338), pMAPK(T202/Y204) (mitogen-activated protein kinase [MAPK] pathway), pS6(S240/244), p70S6k(T389) (mTOR pathway), and pAKT(S473). A novel mechanism for response was discovered whereby high expression level of CAV-1 at the plasma membrane disrupts the BRaf/CRaf heterodimer and thus inhibits the activation of MAPK pathway during dasatinib treatment. Our in vitro and in vivo results provide a new understanding of EphA2 targeting by dasatinib and identify key predictors of therapeutic response. These findings have implications for ongoing dasatinib-based clinical trials.

Nuclear Proteomics with XRCC3 Knockdown to Reveal the Development of Doxorubicin-Resistant Uterine Cancer

The nucleus is a key organelle in mammary cells, which is responsible for several cellular functions including cell proliferation, gene expression, and cell survival. In addition, the nucleus is the primary targets of doxorubicin treatment. In the current study, low-abundance nuclear proteins were enriched for proteomic analysis by using a state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the nuclear protein profiling changes responsible for the development of doxorubicin resistance in human uterine cancer cells. The results of the nuclear proteomic analysis indicated that more than 2100 protein features were resolved from an equal pooled amount of three purified nuclear proteins and 117 differentially expressed spots were identified. Of these 117 identified proteins, 48 belonged to nuclear proteins and a positive correlation was observed between the expression levels of 32 of these nuclear proteins and an increase in drug resistance. According to our review of relevant research, nuclear proteins such as DNA repair protein XRCC3 (XRCC3) have not been reported to play roles in the formation of doxorubicin resistance. Previous studies have used RNA interference and cell viability analysis to evidence the essential roles of XRCC3 on its potency in the formation of doxorubicin resistance. To sum up, our nuclear proteomic approaches enabled us to identify numerous proteins, including XRCC3, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for treating doxorubicin-resistant uterine cancer.

Effect of methotrexate conjugated PAMAM dendrimers on the viability of MES-SA uterine cancer cells.

The aim of this work was to synthesize methotrexate (MTX)-polyamidoamine (PAMAM) dendritic nanoconjugates and to study their effect on cell viability in uterine sarcoma cells. The amide-bonded PAMAM dendrimer-MTX conjugates were prepared by conjugation between the amine-terminated G5 dendrimer and the carboxylic groups of the MTX using a dicyclohexylcarbodiimide coupling reaction. The formation of conjugates was evaluated by ultraviolet (UV) and 1H nuclear magnetic resonance (1H NMR) spectroscopy studies. The cell survival of MES-SA cells, a uterine sarcoma cell line, was evaluated in the presence of the dendrimer-MTX nanoconjugate, using appropriate controls. The UV and 1H NMR study confirmed the formation of covalent bonds between the drug and the dendrimer. The cell viability study indicated that the nanoconjugates had significantly improved cell killing compared to the free MTX.

Effect of a nutrient mixture on matrix metalloproteinase-9 dimers in various human cancer cell lines

Strong clinical and experimental evidence demonstrates association of elevated levels of matrix metalloproteinase MMP-9 with cancer progression, metastasis and shortened patient survival, as it plays a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. MMP-9 is secreted in both the monomeric and dimeric form. Although there is little research on MMP-9 dimers, some studies have shown the dimer to be associated with more aggressive tumor progression. Our objective was to study the relative secretion patterns of MMP-9 monomer and dimer in a variety of cancer cell lines and the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on MMP-9 secretion. The cancer cell lines were grown in their respective media, supplemented with 10% FBS, penicillin (100U/ml) and streptomycin (100µg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0,10, 50, 100, 500 and 1000µg/ml. Parallel sets of cultures were treated with PMA (100ng/ml) for induction of MMP-9. Cell MMP-9 secretion was assayed by gelatinase zymography. MMP-9 dimer secretion patterns of cancer cells fell into different categories. We observed no MMP-9 dimer in prostate DU-145 and PC-3, pancreatic MIA-Pa-Ca2, colon HCT-116, bladder T-24, head and neck FaDu, glioblastoma A-172, T-98 and LN-18 and leukemia HL-60, Jurkat, and Raji cell lines. MMP-dimer secretion only with PMA induction was seen in breast MCF-7 and MDA-MB-231, uterine SK-UT-1, lung A-549, tongue SC-25, melanoma A2058, osteosarcoma U-2OS, rhabdomyosarcoma, fibrosarcoma HT-1080, chondrosarcoma SW-1350 and liposarcoma SW-872. Cervical HeLa and DoTc24510, renal 786-0 and HCC SK-Hep-1 cells exhibited MMP-9 dimer without PMA treatment and increased secretion with PMA treatment. Sarcomas had the highest levels of MMP-9 monomer and dimer with and without PMA among these cancer cell lines. Cervical, uterine and male breast cancer cell lines showed the next highest levels of MMP-9, followed by breast cancer cell lines. Melanoma, renal, lung, head and neck and HCC showed lower levels and prostate, glioblastoma, bladder and leukemia cell lines the lowest. NM showed dose-dependent inhibition of MMP-9 monomer and dimer in all cell lines tested. In conclusion, high MMP-9 and dimer secretion levels correlated with the most aggressive cancer cell lines. NM was effective in inhibiting MMP-9 and dimer secretion in all cell lines tested, suggesting its therapeutic potential as an antimetastatic agent.

Treatment of Cancer-Associated Retinopathy With Rituximab

Cancer-associated retinopathy (CAR) is a rare autoimmune condition associated with various cancers, causing significant visual impairment. Visual symptoms in CAR may or may not correlate with the extent of systemic disease or its response to chemotherapy, and must be addressed separately from the management of systemic malignancy. Steroids have been the mainstay of CAR therapy. Various immunomodulatory therapies have also been described with varying responses, but the overall visual prognosis remains poor. Rituximab is a monoclonal antibody used in the treatment of non-Hodgkin's B-cell lymphoma and many autoimmune disorders. This case report describes a patient with small cell uterine cancer who initially presented with visual impairment associated with CAR. The patient's deteriorating visual symptoms were successfully halted for an extended, clinically meaningful period with rituximab.

Global gene expression analysis in human uterine epithelial cells defines new targets of glucocorticoid and estradiol antagonism.

In preparation for embryo implantation and pregnancy, the uterine epithelium undergoes a genomic and biological transition that mediates adhesion and invasion of the blastocyst. These events resemble an inflammatory response, and the immune system likely takes an active role in the establishment and maintenance of pregnancy. Although glucocorticoids are primary mediators of the immune system, the functional role of glucocorticoid signaling in the uterine epithelium is not well defined. To investigate the dynamic relationship between glucocorticoids and reproductive hormones, we performed whole-genome microarray analysis in a human uterine endometrial cancer cell line (ECC1 cells) treated with the synthetic glucocorticoid dexamethasone (Dex) alone or in combination with estradiol (E₂). Over 10,000 genes were significantly regulated in the presence of Dex and/or E₂. Surprisingly, unique targets of Dex and E₂ together represented the largest group of regulated genes. Ingenuity pathway analysis found both overlapping and independent regulated networks for each hormone. Several hundred genes were found to be coregulated by Dex and E₂, including several that were antagonistically regulated. The effects of glucocorticoids and E₂ are mediated primarily through the glucocorticoid receptor (NR3C1) and estrogen receptor (ESR1), respectively. In silico promoter analysis revealed that NR3C1 and ESR1 response elements are enriched in antagonistically regulated genes, and signaling through these receptors was required for antagonism. Glucocorticoid and E₂ antagonism of target genes may represent a critical junction between the immune system and female reproductive system. Moreover, identification and ontology analysis of glucocorticoid-regulated genes in a uterine epithelial-like cell line suggests that glucocorticoid signaling regulates important biological functions, including immune cell trafficking and embryonic development.

Role of nuclear proteins in doxorubicin-resistance of human uterine cancer cells

Role of nuclear proteins in doxorubicin-resistance of human uterine cancer cells

Role of asparagine synthetase in doxorubicin-induced resistance

Research has shown drug resistance as the major cause of failure of cancer chemotherapy. In this study, doxorubicin-sensitive human uterine cancer cell (hUCC) MES/SA, as well as doxorubicin-resistant hUCC MES/SA-DxR 2μM and MES/SA-DxR 8μM were used. Subsequently, asparagine synthetase (ASNS), a protein that had previously been proposed to be a putative cancer drug target in our laboratory, was silenced by siRNA knockdown to study the mechanism of doxorubicin-induced resistance further. After potent knockdown of ASNS, cell viability in two doxorubicin-resistant cell lines MES/SA-DxR 2μM and MES/SA-DxR 8μM was decreased, as indicated by an MTT cell proliferation assay. By coupling two-dimensional differential gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, proteins that play a vital role in ASNS signaling network and development of doxorubicin-induced resistance were identified. Among all the proteins that we have identified, GRP78 and AKR1C1 appear to be involved in drug resistance, replication factor C appears to participate in DNA repairing, and PP6C is proposed to play a role in cell cycle arrest.

Activated EGFR stimulates MUC1 expression in human uterine and pancreatic cancer cell lines

MUC1 is a large cell surface mucin glycoprotein that plays diverse roles in both normal and tumor cell biology. These roles include mucosal hydration and protection, inhibition of embryo implantation, protection of tumor cells from the immune system and reduction of cytotoxic drug uptake. Similarly, the EGFR family of cell surface receptors drives many normal developmental processes as well as various aspects of tumor growth and gene expression. EGFR family members have been demonstrated to form complexes with MUC1 in various cellular contexts. Nonetheless, the role that EGFR activation plays in modulating MUC1 levels has not been considered. In this study, we demonstrate that activated EGFR drives high level MUC1 expression in multiple cell lines of uterine adenocarcinoma and pancreatic cancer origins. In some cells, addition of exogenous EGFR ligands (EGF or HB-EGF) elevates MUC1 levels while addition of the EGFR tyrosine kinase inhibitor, AG1478, reduces MUC1 levels. The thiazolidinedione, rosiglitazone, previously shown to reduce progesterone-stimulated MUC1 expression, also blocks EGFR ligand-driven MUC1 expression. This activity was observed at relatively high rosiglitazone concentrations (above 10 µM) and appeared to be largely PPARγ independent indicating a novel utility of this drug to reduce mucin-expression in various tumor settings. Collectively, these data demonstrate that: (1) activation of EGFR stimulates MUC1 expression in multiple cellular contexts and (2) it may be possible to develop useful interventions to reduce MUC1 expression as a complementary strategy for tumor therapy.

The anti-proliferative effect of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b) pyran is potentiated via induction of estrogen receptor beta and p21 in human endometrial adenocarcinoma cells

In an effort to develop novel therapeutic agents for endometrial cancer, benzopyran derivatives synthesized at our institute display significant inhibitory activity on cellular growth in uterine cancer cells. The current study was undertaken to demonstrate and explore the estrogen receptor (ER) subtype mediated mechanism of action of benzopyran derivative 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b) pyran (K-1) in human endometrial cancer cells. K-1 competitively inhibited the estradiol binding to human ERα and ERβ and showed growth inhibitory activity in human endometrial Ishikawa, HEC1B and primary endometrial adenocarcinoma cells. Transient transactivation assays carried out in COS-1 cells have demonstrated the diminished ERα-ERE mediated- and induced the ERβ-ERE mediated-transactivation triggered by compound. It also induced ER-mediated transactivation of the cyclin-dependent kinase inhibitor (CDKI) p21WAF-1 in both COS-1 cells and in Ishikawa cells. ERβ inducing effects of compound were blocked by ICI182,780. In endometrial adenocarcinoma cells, it induced ERβ and p21 expression significantly whereas the expression of fos, jun and ERα were significantly reduced. In addition, compound promoted ERα–β heterodimerization as observed in Ishikawa cells. These results demonstrate that the benzopyran compound suppressed the cellular growth via ERβ agonism, induction of p21 and via promoting the ERα–β heterodimerization, in addition to its antagonistic effects exerted on ERα, in human endometrial cancer cells. The study suggests that the dual action of benzopyran molecule may be of significant therapeutic value in ERα/β-positive cases of endometrial cancer.

Development of Image Processing Algorithm for Cytological Diagnosis of Uterine Cervical Cancer Tissue Examination

In this study is relevant to cell area extraction for cytological diagnosis of liquid-based cell examination being used for early diagnosis of uterine cervical cancer by utilizing image processing. As existing cytological diagnosis process has been performed manually by cytotechnologist, the amount of cell image that could be processed was limited. Therefore, in this study, cellular domain extraction through which automatic processing of cytological diagnosis process is enabled was performed and its condition was established depending on the size of cell nucleus based on cytological diagnosis standard of uterine cervical cancer cell. Obtained cell image was processed by hough transform matching with cell image by using matlab through preprocessing phase. As a result, 28 sample images among total 30 sample images were succeeded in finding out target cell and two sample images failed to find it. In the future, modification and verification for such failure case may be required to be performed. It is expected that the result of this study could be utilized for diagnosis process automation of liquid-based cell examination.

The Analysis of Metallothionein Immunoreactivity in Stromal Fibroblasts and Macrophages in Cases of Uterine Cervical Carcinoma With Respect to Both the Local and Distant Spread of the Disease

The tumor microenvironment is made up of tissue that is responsible for the growth and progression of the tumor as well as its ability to initiate metastases. The cancer cells on the front of the tumor together with the macrophages and fibroblasts help to constitute the aggressive phenotype of the tumor. The presence of this aggressive phenotype is indicated by the local infiltration of cancer cells and by the development of lymph node metastases. In cases of uterine cancer, the extent of the local and distant spread of the disease is crucial for determining the type of therapeutic strategy to be applied - surgery alone, surgery followed by radio-chemotherapy, or radio-chemotherapy alone. In the interest of trying to improve the patient's quality of life, different studies supporting the therapeutic model of surgery alone have been conducted. While the cancer cells on the tumor front together with the macrophages and the fibroblasts help to constitute the aggressive phenotype of the tumor, metallothionein (MT) has been shown to have both pro-proliferative and anti-apoptotic activities and to participate in microenvironment remodeling. The aim of the current study was to determine the levels of MT immunoreactivity in the uterine cervical cancer cells as well as in the stromal fibroblasts and macrophages of the tumor microenvironment with respect to the depth of the local invasion and the extent of the distant metastases, so that its potential predictive value as a therapeutic strategy for cervical cancer can be ascertained. We determined the levels of immunoreactivity of MT in a total of 57 carcinoma tissue samples (in the tumor front, in its central part, and in the macrophages and fibroblasts present within the tumor microenvironment). The patients from whom the samples derived were divided into groups with respect to the presence of lymph node metastases (distant spread) and to the depth of invasion (local spread) in relation to the FIGO stage. Metallothionein immunoreactivity was observed in uterine cervical cancer cells; it was also identified in the fibroblasts and macrophages found within the microenvironments of the tumors of patients suffering from the disease. The MT immunoreactivity level significantly increased within the whole cancer nest in relation to the FIGO stage (intensity of the local spread of the disease). Similarly, the infiltration of MT-positive CAFs and TAMs statistically significantly increased in relation to the FIGO stage. The level of MT immunoreactivity found in the fibroblasts and macrophages within the tumor microenvironment seems to be indicative of the intensity of the remodeled cervical tumor microenvironment, and this in turn may be related to the local advancement of the disease. Moreover, it appears that the intensity of the metallothionein immunoreactivity in the immunoreactivity profile of the cervical tumor may be linked to both the depth of the local invasion and the extent of the distant advancement of the disease.

Keratin 8 and 18 Loss in Epithelial Cancer Cells Increases Collective Cell Migration and Cisplatin Sensitivity through Claudin1 Up-regulation

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

Novel MicroRNAs regulating proliferation and apoptosis in uterine papillary serous carcinomas

MicroRNAs (miRNAs) are endogenous, non-coding RNA transcripts that regulate gene expression. Here, we report 175 putative novel miRNAs identified in uterine cancers profiled by Next Generation Sequencing. Our data indicate that one of these putative miRNAs (BCM-173) is conserved across multiple species and is expressed at levels similar to known human miRNAs. Functionally, this miRNA promotes the growth and migration of uterine cancer cell lines by targeting vinculin and altering the distribution of focal adhesions. These results expand our insight into the repertoire of human miRNAs and identify novel pathways by which dysregulated miRNA expression promotes uterine cancer growth.

Enhancement of adriamycin cytotoxicity by sodium butyrate involves hTERT downmodulation‐mediated apoptosis in human uterine cancer cells

Activation of telomerase is a key element in oncogenesis and resistance to apoptosis for many cancers. Some histone deacetylase inhibitors (HDACi) or chemotheraputic agents have been reported to downregulate the expression of human telomerase reverse transcriptase (hTERT). However, whether hTERT is involved in cell death of uterine cancer cells induced by combination of HDACi with chemotheraputic regents remain unknown. The present study shows that combining sodium butyrate (NaBu) and adriamycin (ADR) inhibits proliferation of uterine cancer cell lines in a concentration and time-dependent manner. Growth inhibition was accompanied by caspase-dependent apoptosis with reduced telomerase activity and decreased hTERT mRNA expression. Ectopic wild type (WT)-hTERT suppressed the apoptosis induced by NaBu/ADR treatment, while knockdown of hTERT sensitized uterine cancer cells to ADR. Moreover, the addition of NaBu significantly enhanced ADR cytotoxicity for the primary uterine cancer cells with high hTERT expression. These data indicate that downregulation of hTERT is an important part of the mechanism by which NaBu enhances ADR-induced apoptosis, and suggests that combining NaBu and ADR may be effective in treating uterine tumor with high telomerase activity.

Increasing the rate of heating: A potential therapeutic approach for achieving synergistic tumour killing in combined hyperthermia and chemotherapy

Purpose: A synergistic cancer cell killing effect of sub-lethal hyperthermia and chemotherapy has been reported extensively. In this study, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted in order to investigate the role of heating rate in achieving a synergistic effect. The mode of cell death, induction of thermal tolerance and P-glycoprotein (P-gp) mediated MDR following two different rates of heating were studied.Materials and methods: Doxorubicin (DOX) was used as the chemotherapy drug. A rapid rate hyperthermia was achieved by near infrared laser (NIR) excited indocyanine green (ICG) dye (absorption maximum at 808 nm, ideal for tissue penetration). A slow rate hyperthermia was provided by a cell culture incubator.Results: The potentiating effect of hyperthermia to chemotherapy can be maximised by increasing the rate of heating. When delivered at the same thermal dose, a rapid increase in temperature from 37°C to 43°C caused more cell membrane damage than gradually heating the cells from 37°C to 43°C and thus allowed for more intracellular accumulation of DOX. Meanwhile, the rapid rate laser-ICG hyperthermia at 43°C caused cell necrosis whereas the slow rate incubator hyperthermia at 43°C induced mild apoptosis. At 43°C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified.Conclusions: This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR.

HMMC‐1, a human monoclonal antibody to fucosylated core 1 O‐glycan, suppresses growth of uterine endometrial cancer cells

HMMC-1 is a human monoclonal antibody that reacts with a fucosylated and extended core 1 O-glycan, Fucα1-2Galβ1-4GlcNAcβ1-3Galβ1-3GalNAc-Ser/Thr, as an epitope. In the present study, we examined the effects of HMMC-1 on cell proliferation of two human uterine endometrial cancer cell lines, HEC8 and HEC9, to investigate the role of glycoproteins bearing the HMMC-1 epitope in cancer progression. HEC9 cells expressed high levels of the HMMC-1 epitope, but HMMC-1 reactivity was hardly detected in HEC8 cells. In a mouse model of lymph node metastasis using orthotopic implantation, HEC8 and HEC9 showed low (10%) and high (80%) metastatic potency, respectively. Growth of HEC9, but not HEC8, was remarkably inhibited by addition of HMMC-1 to the culture medium. Cell cycle analysis and expression analysis showed that HMMC-1 treatment increased the G(1) phase population of HEC9 cells and induced cyclin-dependent kinase inhibitors p16 and p21. Two glycoproteins, 97 and 137 kDa, with a strong reactivity to HMMC-1 were purified, and the 97-kDa glycoprotein was identified as CD166, an immunoglobulin superfamily cell adhesion molecule assumed to be involved in cancer metastasis. CD166 gene-silencing dramatically reduced HMMC-1 epitope expression and growth in HEC9 cells, indicating that CD166 is the primary glycoprotein presenting the HMMC-1 epitope in HEC9 cells. Collectively, HMMC-1 might arrest the cell cycle in the G(1) phase by binding to O-glycans on the CD166 expressed in HEC9 cells, raising the possibility that HMMC-1 extensively inhibits invasive growth of HMMC-1 epitope-positive uterine endometrial cancer cells by targeting the cancer-associated form of CD166.

Runx2 Transcription Factor Regulates Heparanase-Induced Bone Resorption in Multiple Myeloma

Abstract 567 Background.Despite advances in treatment strategies, myeloma remains incurable, and bone destruction is a major cause of morbidity in myeloma patients. We have documented in earlier studies that heparanase enzyme is preferentially expressed in myeloma cells and induces severe bone destruction in myeloma. We also discovered that heparanase increases the production of two major bone resorbing factors named Receptor Activator of NF-κB Ligand (RANKL) and Matrix Metalloproteinase 9 (MMP-9) by myeloma cells. Runx2, a member of the runt-related gene family, is a bone-specific transcription factor. Runx2 regulates osteoblast differentiation and is essential for bone tissue development. Interestingly, Runx2 also controls expression of RANKL and MMP-9 genes in osteoblasts. Recent evidence indicates that ectopic induction and overexpression of Runx2 in breast, uterine and prostate cancer cells is associated with bone-metastasis, and osteolytic bone disease in these cancers. However, very little is known about the function of Runx2 in myeloma cells. Here we report for the first time that heparanase engages the Runx2 pathway to promote expression of RANKL and MMP-9 in myeloma cells. Methods.Molecular, biochemical, cellular and in vivo approaches were used to assess the role of Runx2 in heparanase-induced expression of RANKL and MMP-9. These included: (1) Real-time PCR and Western blot analysis to monitor Runx2 levels in CAG myeloma cells expressing high level of heparanase (HPSE-high cells) or with knockdown of endogenous heparanase (HPSE k/d cells), and the corresponding control cells. (2) Chromatin Immunoprecipitation (ChIP) assay to determine in vivo occupancy of the RANKL and MMP-9 gene promoters in myeloma cells by Runx2. (3) Zymography to determine MMP-9 activity in both human and murine myeloma cells. (4) Real-time PCR to determine changes in RANKL and MMP-9 gene expression in Runx2 knockdown MM1.S and 5TGM1 myeloma cells. (5) Assessment of tumor growth/burden and bone resorption in the 5TGM1 syngenic model of murine myeloma. 5TGM1 cells with specific knockdown of Runx2 or non-target shRNA were injected into C57BL/KaLwRij mice through the tail vein and levels of IgG2b and TRAP5b were monitored in the sera of the mice by ELISA. Results.We find Runx2 expression is significantly increased in CAG myeloma cells expressing a high level of heparanase and dramatically reduced in HPSE k/d cells. Increased Runx2 in HPSE-high myeloma cells results in an increase in expression of RANKL and MMP-9 mRNA as well as MMP-9 activity. In sharp contrast, knockdown of Runx2 in human myeloma MM1.S and murine myeloma 5TGM1 cells results in a significant reduction of RANKL, MMP-9 gene expression and MMP-9 activity. Thus heparanase promotes RANKL and MMP-9 expression via Runx2. Analysis of initial 1-kb sequences of RANKL and MMP-9 gene promoter in human and mice, revealed presence of multiple high affinity Runx2-binding sites. ChIP assay confirmed that Runx2 mediates induction of RANKL and MMP-9 gene transcription in the both human and murine myeloma cells by direct association with the proximal promoter of these genes. Together these results demonstrate that Runx2 is a positive regulator of RANKL and MMP-9 gene expression in myeloma cells. Finally, in the syngenic model, knockdown of Runx2 in 5TGM1 murine myeloma cells remarkably inhibits the growth of these cells in vivo and bone resorption. Conclusions.Runx2 is a central regulator of heparanase induced myeloma bone disease. Our discoveries provide new insight into the mechanism of myeloma-induced bone disease and identify Runx2 as a novel target to block myeloma bone disease. Disclosures:No relevant conflicts of interest to declare.

Sequential effects of the proteasome inhibitor bortezomib and chemotherapeutic agents in uterine cervical cancer cell lines

Although the prognosis of uterine cervical cancer has improved due to the advances of treatment modalities, survival of recurrent or metastatic cervical cancer remains poor. Cisplatin is an effective radiosensitizer, but its single agent activity in recurrent cervical cancer is disappointing. Inactivation of tumor suppressors through ubiquitin-mediated degradation by human papillomavirus is known to be a critical step in the carcinogenesis of uterine cervix. Bortezomib, a selective inhibitor of the proteasome, has been shown to inhibit the growth of several solid tumors. To determine the role of bortezomib in cervical cancer as a chemotherapeutic agent, we studied its biological properties. Bortezomib efficiently inhibited the proteasomal activities in cervical cancer cells, and an increased expression of tumor suppressors such as p53, hDlg and hScrib became evident. In addition, sequential or concomitant treatment of bortezomib and cisplatin stimulated the expression of p53, hScrib and p21 and the stimulation was markedly influenced by the order of drugs in HeLa cells. We further confirmed that the concomitant use of bortezomib and cisplatin has synergistic inhibitory effects on the growth of xenograft tumors derived from HeLa cells. Our data establish the possibility that the concomitant use of bortezomib and cisplatin could be an alternative choice in cases resistant to conventional chemotherapy, and sequential effects must be considered for advanced and therapy-resistant cervical cancer patients.