Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.

Correlation of tumor- and stromal-derived MT1-MMP expression with progression of human ovarian tumors in SCID mice

Human ovarian carcinoma samples were orthotopically implanted into SCID mice to investigate the contribution of matrix metalloproteases (MMPs) to the spread of ovarian tumors. Mice were inoculated with patient tumor samples, and developed ovarian tumors over a 16-week period with metastasis occurring in some mice. Species-specific quantitative RT-PCR was used to identify the source of tumor-associated MMPs. Membrane-type (MT)1-MMP mRNA was significantly increased in high-grade tumors, tumors with evidence of serosal involvement, and tumors in which distant metastases were detected. The increase in MT1-MMP expression was predominantly from the human tumor cells, with a minor contribution from the mouse ovarian stroma. Neither human nor mouse MT2-MMP were correlated with tumor progression and MT3-MMP levels were negligible. While tumor cells did not produce significant amounts of MMP-2 or MMP-9, the presence of tumor was associated with increased levels of MMP-2 expression by mouse ovarian stroma. Stromal-derived MT1-MMP was greater in large tumors and was associated with stromal MMP-2 expression but neither was significantly linked with metastasis. These studies indicate that tumor-derived MT1-MMP, more so than other gelatinolytic MMPs, is strongly linked to aggressive tumor behavior. This orthotopic model of human ovarian carcinoma is appropriate for studying ovarian tumor progression, and will be valuable in the further investigation of the metastatic process.

Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

Induction of tumorigenicity by galectin-3 in a nontumorigenic human breast-carcinoma cell-line

The human galectin-3 is a galactoside-binding protein of 31 kDa which functions as a receptor for glycoproteins containing poly N-acetyllactosamine side chains and as a substrate for matrix metalloproteinases-2 and -9. We studied its expression by flow cytofluorimetry, Western, Northern and Southern analyses, in five cultured human breast carcinoma cell lines previously characterized as non-tumorigenic, poorly metastatic or metastatic in nude mice. The expression of galectin-3 correlated with the reported tumorigenicity of the cells. The introduction of recombinant galectin-3 into the null expressing non-tumorigenic BT-549 cells resulted in the acquisition of anchorage-independent growth properties in alland tumorigenicity in 3/4 sense transfected cell crones. The data indicate a relationship between galectin-3 expression and malignancy of human breast carcinoma cell lines.

Expression of β1 integrin in laminin-adhesion-selected human colon cancer cell lines of varying tumorigenicity

Laminin has been shown to promote the malignant phenotype and the expression of certain laminin receptors has been correlated with the malignant character of the tumors. Here new cell lines were isolated from a human colon cancer cell line (LCC-C1) based on their adhesiveness to laminin. The laminin-adherent subclone formed large tumors in nude mice, whereas the laminin-nonadherent subclone failed to form sizable tumors. Only the laminin-adherent subclone adhered to laminin and invaded through Matrigel-coated filters. The adhesive and invasive ability of the cells was almost completely blocked by low concentrations (1.0 μg/ml) of anti-β1 integrin antibody. The amounts of total cellular β1 integrin protein were similar in the two subclones when compared by Western blot, and the mRNA levels also did not differ. The localization of β1 integrin laminin receptor varied in the two subclones; the laminin-adherent subclone showed a linear distribution along the cell-cell junctions, while the laminin-nonadherent subclone did not stain between the cells. Using laminin-Sepharose affinity chromatography, more β1 integrin was obtained from the laminin-adherent subclone. These findings suggest that alterations in the affinity of β1 integrin for laminin and in its membrane distribution might be involved in the increased tumorigenicity observed in colon cancer cells.

Multiplexed tandem polymerase chain reaction identifies strong expression of oestrogen receptor and Her-2 from single, formalin-fixed, paraffin-embedded breast cancer sections

Aim To establish the suitability of multiplex tandem polymerase chain reaction (MT-PCR) for rapid identification of oestrogen receptor (ER) and Her-2 status using a single, formalin-fixed, paraffin-embedded (FFPE) breast tumour section. Methods Tissue sections from 29 breast tumours were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). RNA extracted from 10μm FFPE breast tumour sections from 24 of 29 tumours (14 ER positive and 5 Her-2 positive) was analysed by MT-PCR. After establishing a correlation between IHC and/or FISH and MT-PCR results, the ER/Her-2 status of a further 32 randomly selected, archival breast tumour specimens was established by MT-PCR in a blinded fashion, and compared to IHC/FISH results. Results MT-PCR levels of ER and Her-2 showed good concordance with IHC and FISH results. Furthermore, among the ER positive tumours, MT-PCR provided a quantitative score with a high dynamic range. Threshold values obtained from this data set applied to 32 archival tumour specimens showed that tumours strongly positive for ER and/or Her-2 expression were easily identified by MT-PCR. Conclusion MT-PCR can provide rapid, sensitive and cost-effective analysis of FFPE material and may prove useful as triage to identify patients suited to endocrine or trastuzumab (Herceptin) treatment.

Vimentin expression in cervical carcinomas : association with invasive and migratory potential

Vimentin is an intermediate filament protein normally expressed in mesenchymal cells, but evidence is accumulating in the literature which suggests that the aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastatic potential. Vimentin expression has previously been associated with invasive properties in an in vitro model consisting of a set of HPV-33-transformed cervical keratinocyte cell lines. In the present study, in order to emphasize those in vitro findings, the expression of vimentin has been investigated in cervical neoplasms of different grades, using immunohistochemistry. A clear association is reported between vimentin expression and metastatic progression, since vimentin was detected in all invasive carcinomas and lymph node metastases, but not in CIN III lesions. These in vivo results are compared with present and previous data obtained in vitro on cervical keratinocyte cell lines, where vimentin expression also correlated with in vitro invasiveness.

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase- 2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial- to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4- MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen- induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen- stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.

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.

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell-specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V-X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro-oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

Engineering salinity tolerance in rice by exogenous expression of cell death regulators

Rice, an important crop that feeds more than half of the world's population is very sensitive to salinity stress – a growing problem affecting crop production globally. This PhD study addressed this problem by manipulating the programmed cell death pathways in rice resulting in significant enhancement of salinity stress tolerance. The impact of this work is that farmers would be in a position to grow rice containing such a trait in environments where salinisation of the soil exists, thereby addressing food security needs.

Biological performance of a polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

PURPOSE. We develop a sheep thoracic spine interbody fusion model to study the suitability of polycaprolactone-based scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within the thoracic spine. The surgical approach is a mini- open thoracotomy with relevance to minimally invasive deformity correction surgery for adolescent idiopathic scoliosis. To date there are no studies examining the use of this biodegradable implant in combination with biologics in a sheep thoracic spine model. METHODS. In the present study, six sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone-based scaffold plus 0.54μg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion was assessed at six months post-surgery. RESULTS. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL- based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluations of the respective groups. Biomechanical testing revealed significantly higher stiffness for the rhBMP-2 plus PCL- based scaffold group in all loading directions in comparison to the other two groups. CONCLUSION. The results of this study demonstrate that rhBMP-2 plus PCL- based scaffold is a viable bone graft substitute, providing an optimal environment for thoracic interbody spinal fusion in a large animal model.

6 versus 12 month performance of polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

Introduction Well-designed biodegradable scaffolds in combination with bone growth factors offer a valuable alternative to the current gold standard autograft in spinal fusion surgery Yong et al. (2013). Here we report on 6- vs 12- month data set evaluating the longitudinal performance of a CaP coated polycaprolactone (PCL) scaffold loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within a large preclinical animal model. Methods Twelve sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone based scaffold plus 0.54µg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion assessments were performed via high resolution clinical computed tomography and histological evaluation were undertaken at six (n=6) and twelve (n=6) months post-surgery using the Sucato grading system (Sucato et al. 2004). Results The computed tomography fusion grades of the 6- and 12- months in the rhBMP-2 plus PCL- based scaffold group were 1.9 and 2.1 respectively, in the autograft group 1.9 and 1.3 respectively, and in the scaffold alone group 0.9 and 1.17 respectively. There were no statistically significant differences in the fusion scores between 6- and 12- month for the rhBMP plus PCL- based scaffold or PCL – based scaffold alone group however there was a significant reduction in scores in the autograft group. These scores were seen to correlate with histological evaluations of the respective groups. Conclusions The results of this study demonstrate the efficacy of scaffold-based delivery of rhBMP-2 in promoting higher fusion grades at 6- and 12- months in comparison to the scaffold alone or autograft group within the same time frame. Fusion grades achieved at six months using PCL+rhBMP-2 are not significantly increased at twelve months post-surgery.

PRMT2 and RORγ expression are associated with breast cancer survival outcomes

Protein arginine methyltransferases (PRMTs) methylate arginine residues on histones and target transcription factors that play critical roles in many cellular processes, including gene transcription, mRNA splicing, proliferation, and differentiation. Recent studies have linked PRMT-dependent epigenetic marks and modifications to carcinogenesis and metastasis in cancer. However, the role of PRMT2-dependent signaling in breast cancer remains obscure. We demonstrate PRMT2 mRNA expression was significantly decreased in breast cancer relative to normal breast. Gene expression profiling, Ingenuity and protein-protein interaction network analysis after PRMT2-short interfering RNA transfection into MCF-7 cells, revealed that PRMT2-dependent gene expression is involved in cell-cycle regulation and checkpoint control, chromosomal instability, DNA repair, and carcinogenesis. For example, PRMT2 depletion achieved the following: 1) increased p21 and decreased cyclinD1 expression in (several) breast cancer cell lines, 2) decreased cell migration, 3) induced an increase in nucleotide excision repair and homologous recombination DNA repair, and 4) increased the probability of distance metastasis free survival (DMFS). The expression of PRMT2 and retinoid-related orphan receptor-γ (RORγ) is inversely correlated in estrogen receptor-positive breast cancer and increased RORγ expression increases DMFS. Furthermore, we found decreased expression of the PRMT2-dependent signature is significantly associated with increased probability of DMFS. Finally, weighted gene coexpression network analysis demonstrated a significant correlation between PRMT2-dependent genes and cell-cycle checkpoint, kinetochore, and DNA repair circuits. Strikingly, these PRMT2-dependent circuits are correlated with pan-cancer metagene signatures associated with epithelial-mesenchymal transition and chromosomal instability. This study demonstrates the role and significant correlation between a histone methyltransferase (PRMT2)-dependent signature, RORγ, the cell-cycle regulation, DNA repair circuits, and breast cancer survival outcomes.

Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.