Anne E. Cress

Role for DNA Methylation in the Regulation of miR-200c and miR-141 Expression in Normal and Cancer Cells

Background The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood. Methodology/Principal Findings Epigenetic mechanisms participate in the control of miR-200c and miR-141 expression in both normal and cancer cells. A CpG island near the predicted mir-200c/mir-141 transcription start site shows a striking correlation between miR-200c and miR-141 expression and DNA methylation in both normal and cancer cells, as determined by MassARRAY technology. The CpG island is unmethylated in human miR-200/miR-141 expressing epithelial cells and in miR-200c/miR-141 positive tumor cells. The CpG island is heavily methylated in human miR-200c/miR-141 negative fibroblasts and miR-200c/miR-141 negative tumor cells. Mouse cells show a similar inverse correlation between DNA methylation and miR-200c expression. Enrichment of permissive histone modifications, H3 acetylation and H3K4 trimethylation, is seen in normal miR-200c/miR-141-positive epithelial cells, as determined by chromatin immunoprecipitation coupled to real-time PCR. In contrast, repressive H3K9 dimethylation marks are present in normal miR-200c/miR-141-negative fibroblasts and miR-200c/miR-141 negative cancer cells and the permissive histone modifications are absent. The epigenetic modifier drug, 5-aza-2′-deoxycytidine, reactivates miR-200c/miR-141 expression showing that epigenetic mechanisms play a functional role in their transcriptional control. Conclusions/Significance We report that DNA methylation plays a role in the normal cell type-specific expression of miR-200c and miR-141 and this role appears evolutionarily conserved, since similar results were obtained in mouse. Aberrant DNA methylation of the miR-200c/141 CpG island is closely linked to their inappropriate silencing in cancer cells. Since the miR-200c cluster plays a significant role in EMT, our results suggest an important role for DNA methylation in the control of phenotypic conversions in normal cells.

N-Cadherin Expression in Human Prostate Carcinoma Cell Lines : An Epithelial-Mesenchymal Transformation Mediating Adhesion with Stromal Cells

In human prostate adenocarcinoma, an association between loss of E-cadherin, increased Gleason score, and extracapsular dissemination has been observed. Further characterization of the E-cadherin/catenin phenotype of human prostate carcinoma cell lines showed loss of E-cadherin and expression of N-cadherin in poorly differentiated prostate carcinoma cell lines (PC-3N derived from PC-3, PC-3, and JCA1). We showed that N-cadherin is concentrated at sites of cell-cell contact in PC-3N cellular extensions. N-cadherin was also expressed in prostate stromal fibroblasts both in vitro and in prostate tissue. Co-cultures of prostate stromal fibroblasts and PC-3N cells showed the immunolocalization of N-cadherin in intercellular contacts. In addition, the isoform expression of the cadherin binding protein p120(ctn) differed in relation to the expression of E- versus N-cadherin by the prostate carcinoma cell lines. The p100 isoform was more highly expressed in E-cadherin-positive carcinoma cell lines, whereas p120 was predominantly expressed only in N-cadherin-positive prostate carcinoma cell lines and prostate stromal fibroblasts. The N-cadherin-positive carcinoma cell line, PC-3N, displayed aggressive invasion into the surface of the diaphragm muscle after intraperitoneal injection of SCID mice. The gain of N-cadherin and loss of E-cadherin by invasive prostate carcinoma cell lines suggests a progression from an epithelial to a mesenchymal phenotype, which may allow for their interaction with surrounding stromal fibroblasts and facilitate metastasis.

The α6β1 and α6β4 integrins in human prostate cancer progression

SummaryProstatic secretions are formed by glands composed of basal and luminal cells and surrounded by a basal lamina. The normal basal cells express several integrins (extracellular matrix receptors) including alpha 2, 3, 4, 5, 6, v, beta 1 and beta 4. These integrin units are polarized at the base of the cells adjacent to the basal lamina. The integrin alpha 6 beta 4 is associated with hemidesmosomal-like structures.The natural history of prostate cancer involves the presence of prostatic intraepithelial neoplasia (PIN) lesions (considered precursor lesions), carcinomain situ and invasive carcinoma. Hemidesmosomal proteins and the α3β1 and α6β1 integrins (laminin receptors) are retained in the early PIN lesions. Expression of the integrins α2, α4, α5, αv and β4 is lost in carcinoma. The α3β1 and α6β1 integrins remain associated with invasive carcinoma, the latter being predominant. Integrin expression in carcinoma is diffuse in the plasma membrane and not restricted to the basal aspects of the cell. The α6β1 integrin is fully functional as judged by an ability to adhere to laminin and contains the wild type α6A cytoplasmic signaling domain. The α6β1 integrin is a leading candidate for conferring the invasive phenotype in prostatic carcinoma.Tumor cells with high expression of α6 integrin are more invasive when tested in a SCID mouse model system. Following intraperitoneal injection, the human tumor cells invade the mouse diaphragm and move through the muscle on the surface of the laminin coated muscle cells. Our current working hypothesis is that the production of α6β1 and laminin in human tumor cells contributes to the invasive phenotype. Invasion could occur on the surfaces of laminin coated structures such as the nerves, blood vessels or muscle and account for the known patterns of human prostate tumor progression. Blockage of the expression or function of α6β1 or laminin or preventing the loss of β4 would be essential steps in confining the carcinoma to the prostate gland where conventional treatment has already proven effective.

Integrin α6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotype in vitro and in vivo

Cell adhesion and migration are important features in tumor invasion, being mediated in part by integrins (extracellular matrix receptors). Integrins are significantly decreased in human prostate cancer. An exception is α6 integrin (laminin receptor) which persists during prostate tumor progression. We have selected high (DU-H) and low (DU-L) expressors of α6 integrin from a human prostate tumor cell line, DU145, to assess experimentally the importance of α6 integrin in tumor invasion. DU-H cells exhibited a four-fold increased expression of α6 integrin on the surface compared to DU-L cells. Both cell types contained similar amounts of α3 and α5 integrin. The DU-H cells contained α6 subunits complexed with both the β1 and β4 subunits whereas DU-L cells contained α6 complexed only with β4. DU-H cells were three times more mobile on laminin as compared to DU-L, but adhered similarly on laminin. Adhesion and migration were inhibited with anti-α6 antibody. Each subline was injected intraperitoneally into SCID mice to test its invasive potential. Results showed greater invasion of DU-H compared to DU-L cells, with increased expression of a6 integrin on the tumor at the areas of invasion. These data suggest that α6 integrin expression is advantageous for prostate tumor cell invasion.

Characterization of integrin subunits, cellular adhesion and tumorgenicity of four human prostate cell lines

Cellular adhesion to extracellular matrix proteins via integrin molecules is a major factor in the process of invasion and metastasis of human tumor cells. Four human prostate cell lines were characterized according to the presence and quantity of integrin subunits, the ability of the cells to attach to extracellular substrates and the capacity of the cells to form tumors in severe combined immunodeficient (SCID) mice. All four human prostate cell lines expressed three to five integrins on their cell surfaces. The DU145, PC3 and 431P cells expressed primarily α3, α5, and α6 integrin at similar levels. These cell lines expressed the subunits β1, β3 and β4 with β1 predominant. The DU145 cells preferred attachment to fibronectin, followed by laminin and vitronectin. Approximately 50%–60% of the binding of DU145 cells to fibronectin and laminin was dependent on the function of α5β1 and α6 respectively. The cell line LNCaP differed in its low expression of the α3 subunit, 95% of cellular adhesion to fobronectin and laminin being integrin-dependent and its inability to attach to vitronectin, in spite of surface expression of αvβ3. All the cell lines except for LNCaP readily formed tumors within SCID mice and the expression of α3, α6, β1, and β4 integrin subunits was preserved in the resulting tumor tissue. The altered adhesion properties of the LNCaP cells may explain their altered tumorigenicity.

Unique expression pattern of the α6β4 integrin and laminin-5 in human prostate carcinoma

The α6β4 integrin and its ligand, laminin‐5, are essential gene products for the maintenance and remodeling of a stratified epithelium. Apparent loss of polarized α6β4 integrin and laminin‐5 protein expression in invasive prostate cancer as compared to normal prostate glands is known to occur. It is unknown whether these alterations occur in prostatic intraepithelial neoplasia (PIN) lesions and whether this combined defect occurs in other epithelial cancers.

Epigenetic silencing of DSC3 is a common event in human breast cancer

IntroductionDesmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.MethodsWe used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines.ResultsDSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure.ConclusionThese results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

Morphine treatment accelerates sarcoma-induced bone pain, bone loss, and spontaneous fracture in a murine model of bone cancer

Abstract Metastatic bone cancer causes severe pain that is primarily treated with opioids. A model of bone cancer pain in which the progression of cancer pain and bone destruction is tightly controlled was used to evaluate the effects of sustained morphine treatment. In cancer‐treated mice, morphine enhanced, rather than diminished, spontaneous, and evoked pain; these effects were dose‐dependent and naloxone‐sensitive. SP and CGRP positive DRG cells did not differ between sarcoma or control mice, but were increased following morphine in both groups. Morphine increased ATF‐3 expression only in DRG cells of sarcoma mice. Morphine did not alter tumor growth in vitro or tumor burden in vivo but accelerated sarcoma‐induced bone destruction and doubled the incidence of spontaneous fracture in a dose‐ and naloxone‐sensitive manner. Morphine increased osteoclast activity and upregulated IL‐1β within the femurs of sarcoma‐treated mice suggesting enhancement of sarcoma‐induced osteolysis. These results indicate that sustained morphine increases pain, osteolysis, bone loss, and spontaneous fracture, as well as markers of neuronal damage in DRG cells and expression of pro‐inflammatory cytokines. Morphine treatment may result in “add‐on” mechanisms of pain beyond those engaged by sarcoma alone. While it is not known whether the present findings in this model of osteolytic sarcoma will generalize to other cancers or opioids, the data suggest a need for increased understanding of neurobiological consequences of prolonged opioid exposure which may allow improvements in the use of opiates in the effective management of cancer pain.

The use of a combinatorial library method to isolate human tumor cell adhesion peptides

SummaryTumor cell progression is dependent in part on the successful adhesive interactions of the cells with the extracellular matrix. In this study, a new approach is described to isolate linear peptide ligand candidates involved in cellular adhesion. A synthetic combinatorial peptide library based on the ‘one-bead-one-peptide’ concept was incubated with live human prostate cancer cells for 90 min at 37 °C. The peptide bead coated with a monolayer of cells was then isolated for micro sequencing. The DU145 (DU-H) cells were chosen since they have been previously characterized as containing elevated levels of a laminin receptor for cell adhesion, the α6β1 integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are α6-specific ligand candidates. From two different libraries (linear 9-mer and 11-mer) of a total of 1 500 000 beads, 68 peptide beads containing attached cells were isolated. These positive beads were then retested to determine the ability of the GoH3 antibody to block binding of the cells to the peptide beads. The α6 integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVSVNGRHX) and RX-1 (DNRIRLQAKXX), resemble the previously reported active peptide sequences (GD-2 and AG-73) from native laminin. The RU-1, RX-1 and AG-73 peptides were tested for their ability to support cell attachment and to bind the cell surface of DU-H prostate carcinoma cells in suspension using fluorescence-activated cell-sorting (FACS) analysis. Both RU-1 and AG-73 peptides supported cellular attachment within 1 h. In contrast, after 1 h, EHS laminin supported both cellular attachment and spreading. The RX-1 peptide exhibited only weak binding to the DU-H prostate carcinoma cells. FACS analysis indicated that AG-73 peptide attached to tumor cell surfaces over a range of concentrations, whereas the RU-1 peptide showed a homogeneous concentration required for attachment. The described strategy for screening a random peptide library offers three advantages: (i) ligands for conformationally sensitive receptors of adhesion can be isolated using live cells; (ii) specific binding can be selected for using function-blocking antibodies; and (iii) peptides supporting adhesion independent of spreading properties can be distinguished. In principle, specific adhesive peptides without prior knowledge of the sequence could be isolated for any epithelial cell surface receptor for which a function-blocking reagent is available.

FACTORS REGULATING MEMBRANE PERMEABILITY ALTER THERMAL RESISTANCE

Elevated temperatures (>37OC) produce a number of biological effects that have great potential in the treatment of human malignancies. These include the following: (a) tumor cells may be selectively killed over certain temperature ranges,’** (b) heat interacts synergistically with radiation,’ (c) heat may be most effective at killing cells resistant to radiation, such as !%phase4*’ and hypoxic cells,6.’ and (d) heat may enhance the effects of chemotherapeutic drugs, either by increasing cellular uptake of the drug: by affecting interaction with target molecules, or by affecting cellular repair mechanisms.’ Hyperthermic exposures are known also to induce a transient, nonheritable resistance to subsequent heat e x p o s ~ r e s . ’ ~ ’ ~ It is now being recognized that the phenomena of thermotolerance may be both a hazard, in the case of tumor cells becoming resistant, or an advantage, in the situation where normal cells may become more resistant than tumor cells. It is known that thermotolerance can be induced, either by short exposures (minutes) during fractionated heat doses,’”’* or after long, continuous exposures (3 to 5 hours) to temperatures less than 43°C.3.’3 This resistance has been observed in a wide variety of cell lines, including cells of human,’0*” hamster,” and murine’ origin. Henle and Dethlefson14 have recently reviewed extensively the known phenomenology of thermotolerance, detailing evidence for this effect both in vilro and in vivo. In spite of the extensive knowledge of the temperature, time, and culture condition parameters that affect thermal resistance, the mechanism(s) by which this cellular response to heat develops is poorly understood. This knowledge is essential, however, if thermotolerance is to be manipulated by oncologists in a manner that will be of therapeutic advantage, especially in the area of overcoming resistance once it has developed. Gould and Dring have presented a model,” with supporting evidence,I6 to explain heat resistance in bacterial endospores. This model suggests that the cortex of these organisms acts as a specialized osmoregulatory organelle. They speculate that heat resistance arises from dehydration of the protoplast resulting from osmotic activity of an expanded electronegative polymer and the positively charged ions associated with it. One aspect of this model is that, in the heat-resistant form, the coat of these bacterial endospores is impermeable to multivalent cations, including calcium