Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

LCC15-MB : a vimentin-positive human breast cancer cell line from a femoral bone metastasis

The LCC15-MB cell line was established from a femoral bone metastasis that arose in a 29-year-old woman initially diagnosed with an infiltrating ductal mammary adenocarcinoma. The tumor had a relatively high (8%) S-phase fraction and 1/23 positive lymph nodes (LN). Both the primary tumor and LN metastasis were positive for estrogen receptor (ER) and progesterone receptor (PgR), but lacked erbB2 expression. Approximately one year later, the patient presented with a 0.8 cm comedo-type intraductal mammary adenocarcinoma in the left breast that was negative for ER and PgR, but positive for erbB2. Thirty-five months after the initial diagnosis she was treated for acute skeletal metastasis, and stabilized with a hip replacement. At this time, tumor cells were removed from surplus involved bone, inoculated into cell culture, and developed into the LCC15-MB cell line. The bone metastasis was a poorly differentiated adenocarcinoma lacking ER, PgR, and erbB2, characteristics shared by the LCC15-MB cells, although ER can be re-expressed by treatment of the LCC15-MB cells for 5 days with 75 μM 5-aza-2'-deoxycytidine. The LCC15-MB cell line is tumorigenic when implanted subcutaneously in NCr nu/nu mice and produces long-bone metastases after intracardiac injection. Although the bone metastasis from which the LCC15-MB cell line was derived lacked vimentin (VIM) expression, the original primary tumor and lymph node metastasis were strongly VIM positive, as are LCC15-MB cells in vitro and in nude mice. The karyotype and isozyme profiles of LCC15-MB cells are consistent with its origin from a human female, with most chromosome counts in the hypertriploid range. Thirty-two marker chromosomes are present. These cells provide an in vitro/in vivo model in which to study the inter-relationships between ER, VIM, and bone metastasis in human breast cancer.

Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44 : correlation with invasive potential

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile

Epithelial mesenchymal transition (EMT) and cancer stem cells (CSC) have been associated with resistance to chemotherapy. Eighty percent of ovarian cancer patients initially respond to platinum-based combination therapy but most return with recurrence and ultimate demise. To better understand such chemoresistance we have assessed the potential role of EMT in tumor cells collected from advanced-stage ovarian cancer patients and the ovarian cancer cell line OVCA 433 in response to cisplatin in vitro. We demonstrate that cisplatin-induced transition from epithelial to mesenchymal morphology in residual cancer cells correlated with reduced E-cadherin, and increased N-cadherin and vimentin expression. The mRNA expression of Snail, Slug, Twist, and MMP-2 were significantly enhanced in response to cisplatin and correlated with increased migration. This coincided with increased cell surface expression of CSC-like markers such as CD44, α2 integrin subunit, CD117, CD133, EpCAM, and the expression of stem cell factors Nanog and Oct-4. EMT and CSC-like changes in response to cisplatin correlated with enhanced activation of extracellular signal-regulated kinase (ERK)1/2. The selective MEK inhibitor U0126 inhibited ERK2 activation and partially suppressed cisplatin-induced EMT and CSC markers. In vivo xenotransplantation of cisplatin-treated OVCA 433 cells in zebrafish embryos demonstrated significantly enhanced migration of cells compared to control untreated cells. U0126 inhibited cisplatin-induced migration of cells in vivo, suggesting that ERK2 signaling is critical to cisplatin-induced EMT and CSC phenotypes, and that targeting ERK2 in the presence of cisplatin may reduce the burden of residual tumor, the ultimate cause of recurrence in ovarian cancer patients.

The biology of breast tumor progression : acquisition of hormone independence and resistance to cytotoxic drugs

Many breast tumors appear to follow a predictable clinical pattern, being initially responsive to endocrine therapy and to cytotoxic chemotherapy but ultimately exhibiting a phenotype resistant to both modalities. Using the MCF-7 human breast cancer cell line as an example of an 'early' phenotype (estrogen and progesterone receptor positive, steroid responsive, low metastatic potential), we have isolated and characterized a series of hormone-independent but hormone-responsive variants (MIII and MCF7/LCC1). However, these variants remain responsive to both antiestrogens and cytotoxic drugs (methotrexate and colchicine). MIII and MCF7/LCCl cells appear to mimic some of the critical aspects of the early progression to a more aggressive phenotype. An examination of the phenotype of these cells suggests that some hormone-independent breast cancer cells are derived from hormone-dependent parental cells. The development of a hormone-independent phenotype can arise independently of acquisition of a cytotoxic drug resistant phenotype.

Assays for the study of human cancer cell invasion and metastasis

Two in vitro and two in vivo assays for the study of human cancer invasion and metastasis are described. The assays include in vitro invasiveness through an artificial basement membrane (Matrigel®), invasiveness and metastasis in nude mice of subcutaneously injected LacZ-transduced human tumor cells, in vitro adherence to basement membrane components, and LacZ-transduced human cancer cells injected intravenously into nude mice. In studies of the processes involved in human cancer cell invasion and metastasis, these four assays were found to be complementary, and thus provide a set of test systems for preclinical screening of agents which interfere with these processes.

Epithelial-to-mesenchymal transitions and circulating tumor cells

Epithelial-to-mesenchymal transition (EMT) phenomena endow epithelial cells with enhanced migratory and invasive potential, and as such, have been implicated in many physiological and pathological processes requiring cell migration/invasion. Although their involvement in the metastatic cascade is still a subject of debate, data are accumulating to demonstrate the existence of EMT phenotypes in primary human tumors, describe enhanced metastatic potential of EMT derivatives in animal models, and report EMT attributes in circulating tumor cells (CTCs). The relationships between EMT and CTCs remain largely unexplored, and we review here in vitro and in vivo data supporting a putative role of EMT processes in CTC generation and survival.

Tumor cells are the source of osteopontin and bone sialoprotein expression in human breast cancer

Bone sialoprotein (BSP) and osteopontin (OPN) are secreted glycoproteins with a conserved Arg-Gly-Asp (RGD) integrin-binding motif and are expressed predominantly in bone. The RGD tripeptide is commonly present in extracellular attachment proteins and has been shown to mediate the attachment of osteosarcoma cells and osteoclasts. To determine the origin and incidence of BSP and OPN mRNA expression in primary tumor, a cohort of archival, primary invasive breast carcinoma specimens was analyzed. BSP transcripts were detected in 65% and OPN transcripts in 77% of breast cancers examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages. Despite its abundance in carcinomas, BSP expression was not detected in a panel of 11 human breast cancer cell lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB- 231, MDA-MB-436, BT549, MCF-7(AOR), Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts. Our data suggest that in human primary breast tumors, the origin of BSP and OPN mRNA is predominantly the breast cancer cells and that expression of these transcripts is influenced by the tumor environment.

Radiosensitizing effect of electrochemotherapy in a fractionated radiation regimen in radiosensitive murine sarcoma and radioresistant adenocarcinoma tumor model

Radiosensitizing Effect of Electrochemotherapy in a Fractionated Radiation Regimen in Radiosensitive Murine Sarcoma and Radioresistant Adenocarcinoma Tumor Model. Electrochemotherapy can potentiate the radiosensitizing effect of bleomycin, as shown in our previous studies. To bring this treatment closer to use in clinical practice, we evaluated the interaction between electrochemotherapy with bleomycin and single-dose or fractionated radiation in two murine tumor models with different histology and radiosensitivity. Radiosensitive sarcoma SA-1 and radioresistant adenocarcinoma CaNT subcutaneous tumors grown in A/J and CBA mice, respectively, were used. The anti-tumor effect and skin damage around the treated tumors were evaluated after electrochemotherapy with bleomycin alone or combined with single-dose radiation or a fractionated radiation regimen. The anti-tumor effectiveness of electrochemotherapy was more pronounced in SA-1 than CaNT tumors. In both tumor models, the tumor response to radiation was not significantly influenced by bleomycin alone or by electroporation alone. However, electrochemotherapy before the first tumor irradiation potentiated the response to a single-dose or fractionated radiation regimen in both tumors. For the fractionated radiation regimen, normal skin around the treated tumors was damaged fourfold less than for the single-dose regimen. Electrochemotherapy prior to single-dose irradiation induced more damage to the skin around the treated tumors and greater loss of body weight compared to other irradiated groups, whereas electrochemotherapy combined with the fractionated radiation regimen did not. Electrochemotherapy with low doses of bleomycin can also be used safely for radiosensitization of different types of tumors in a fractionated radiation regimen, resulting in a good anti-tumor effect and no major potentiating effect on radiation-induced skin damage. © 2009 by Radiation Research Society.

Effect of atmospheric gas plasmas on cancer cell signaling

Cancer is one of the most life-threatening diseases with many forms still regarded as incurable. The conventional cancer treatments have unwanted side effects such as the death of normal cells. A therapy that can accurately target and effectively kill tumor cells could address the inadequacies of the available therapies. Atmospheric gas plasmas (AGP) that are able to specifically kill cancerous cells offer a promising alternative approach compared to conventional therapies. AGP have been shown to exploit tumor-specific genetic defects and a recent trial in mice has confirmed its antitumor effects. The mechanism by which the AGP act on tumor cells but not normal cells is not fully understood. A review of the current literature suggests that reactive oxygen species (ROS) generated by AGP induce death of cancer cells by impairing the function of intracellular regulatory factors. The majority of cancer cells are defective in tumor suppressors that interfere normal cell growth pathways. It appears that pro-oncogene or tumor suppressor-dependent regulation of antioxidant/or ROS signaling pathways may be involved in AGP-induced cancer cell death. The toxic effects of ROS are mitigated by normal cells by adjustment of their metabolic pathways. On the other hand, tumor cells are mostly defective in several regulatory signaling pathways which lead to the loss of metabolic balance within the cells and consequently, the regulation of cell growth. This review article evaluates the impact of AGP on the activation of cellular signaling and its importance for exploring mechanisms for safe and efficient anticancer therapies.

Atmospheric gas plasma–induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis

Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity. However, the mechanism of AGP-induced cancer cell death is unknown. In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells. By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells. TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis. Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis. Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis. The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

Targeted disruption of the JAK2/STAT3 pathway in combination with systemic administration of paclitaxel inhibits the priming of ovarian cancer stem cells leading to a reduced tumor burden

Chemotherapy resistance associated with recurrent disease is the major cause of poor survival of ovarian cancer patients. We have recently demonstrated activation of the JAK2/STAT3 pathway and the enhancement of a cancer stem cell (CSC)-like phenotype in ovarian cancer cells treated in vitro with chemotherapeutic agents. To elucidate further these mechanisms in vivo,we used a two-tiered paclitaxel treatment approach in nude mice inoculated with ovarian cancer cells. In the first approach, we demonstrate that a single intraperitoneal administration of paclitaxel in mice 7 days after subcutaneous transplantation of the HEY ovarian cancer cell line resulted in a significant increase in the expression of CA125, Oct4, and CD117 in mice xenografts compared to control mice xenografts which did not receive paclitaxel. In the second approach, mice were administered once weekly with paclitaxel and/or a daily dose of the JAK2-specific inhibitor, CYT387, over 4weeks. Mice receiving paclitaxel only demonstrated a significant decrease in tumor volume compared to control mice. At the molecular level, mouse tumors remaining after paclitaxel administration showed a significant increase in the expression of Oct4 and CD117 coinciding with a significant activation of the JAK2/STAT3 pathway compared to control tumors. The addition of CYT387 with paclitaxel resulted in the suppression of JAK2/STAT3 activation and abrogation of Oct4 and CD117 expression in mouse xenografts. This coincided with significantly smaller tumors in mice administered CYT387 in addition to paclitaxel, compared to the control group and the group of mice receiving paclitaxel only. These data suggest that the systemic administration of paclitaxel enhances Oct4- and CD117-associated CSC-like marker expression in surviving cancer cells in vivo, which can be suppressed by the addition of the JAK2-specific inhibitor CYT387, leading to a significantly smaller tumor burden. These novel findings have the potential for the development of CSC-targeted therapy to improve the treatment outcomes of ovarian cancer patients.

Speckle-type POZ protein mutations interrupt tumor suppressor function of speckle-type POZ protein in prostate cancer by affecting androgen receptor degradation

Large scale exome sequencing studies have revealed regions of the genome, which contribute to the castrate resistant prostate cancer (CRPC) phenotype. [1],[2],[3] Such studies have identified mutations in genes, which may have diagnostic/prognostic potential, or which may be targeted therapeutically. Two of these genes include the androgen receptor (AR) and speckle-type POZ protein (SPOP) genes. However, the findings from these exome sequencing studies can only be translated therapeutically once the functional consequences of these mutations have been determined. Here, we highlight the recent study by An et al. [4] which investigated the functional effects of mutations in the SPOP gene that were identified in the aforementioned exome sequencing studies, particularly in the context of SPOP-mediated degradation of the AR.

Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair

Rationale: Chronic lung disease characterized by loss of lung tissue,inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. Objectives: To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell–like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Methods: Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. Measurements and Main Results: hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A–D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks posttransplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-a, IL-1 and -6, and profibrotic transforming growth factor-b in mouse lungs. In addition,lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors f matrix metalloproteinase-1 and 2. Conclusions: hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.