Haakon Skogseth

A New Method to Provide a Fresh Frozen Prostate Slice Suitable for Gene Expression Study and MR Spectroscopy

Fresh frozen tissue from radical prostatectomy specimens is highly valuable material for research on gene expression and cellular metabolites. The purpose of this study was to develop a standardized method to provide a representative high quality research sample from radical prostatectomy specimens without interfering with the routine histopathological procedure.

Inhibitors of tyrosine kinase inhibit the production of urokinase plasminogen activator in human prostatic cancer cells

Urokinase‐type plasminogen activator (uPA) seems to be an important protease in prostate cancer invasion, and tyrosine phosphorylation is thought to play a role in the regulation of its production. The amount of uPA was measured with a synthetic peptide substrate after treatment with various concentrations of tyrosine kinase inhibitors (TKI). The effect on proliferation and apoptosis was also assayed. Non‐toxic levels of genistein or the tyrphostin AG 490 produced up to 50% reduction of the uPA production in PC‐3 and DU‐145. The tyrphostins AG 1296 and AG 1478 inhibited uPA production in PC‐3 cells, whereas DU‐145 showed a slight increase of uPA production. TKI neither induced any detectable apoptosis, nor was there any reduction in proliferation rate. TKI can profoundly modify the production of uPA in prostatic cancer cells, thus indicating their possible use as suppressors of the invasive phenotype. The therapeutic potential of TKI warrants further investigation.

The invasive behaviour of prostatic cancer cells is suppressed by inhibitors of tyrosine kinase.

Proteolytic enzymes, and especially urokinase plasminogen activator (uPA), play an important role in tumour invasion and metastasis. Previously we demonstrated that the production of urokinase plasminogen activator (uPA) was decreased by several tyrosine kinase inhibitors (TKI) in two prostatic carcinoma cell lines. The effect of the two TKI genistein and tyrphostin AG‐1478 was investigated in the prostate carcinoma cell lines PC‐3 and DU‐145. A reconstituted basal lamina (Matrigel) was used as a migration barrier. The production of matrix metalloproteinases (MMP) was also measured. Roles of plasminogen and uPA were examined. Cell invasion was increased by plasminogen, but this enhanced cell migration was counteracted by TKI treatment. The increased cell invasion induced by plasminogen was decreased by at least 60% in both cell lines when α‐2 anti‐plasmin was added to the assay. Cells in the absence of plasminogen were not affected by TKI. External uPA failed to regenerate the decreased cell invasion caused by TKI. The production of MMP was inhibited by both TKI. Our results indicate a possible role of TKI as inhibitors of cancer cell invasion by inhibiting uPA and MMP production.

Gene expressional changes in prostate fibroblasts from cancerous tissue.

Prostate cancer is the most common type of cancer in men. It is assumed that the tumor microenvironment of the prostate contributes to invasion and metastasis. Stroma‐epithelial crosstalk has shown to change with progression of prostate cancer, and thereby the stromal compartment might be an attractive target in diagnostic and therapeutic approaches to prostate cancer. The purpose of this project was to study the reciprocal influence between fibroblasts and cancer cells in prostate cancer. Prostate fibroblast primary cultures from areas with cancer and hyperplasia were cocultivated with cells of the PC‐3 lineage. Gene expression profiles of both cell types were studied to reveal possible associations to cancer invasion and metastasis. There were 383 differentially expressed genes between fibroblasts from cancerous areas and fibroblasts from areas with hyperplasia before cocultivation with PC‐3 cells. Several of the differentially expressed gene classes are associated with cancer development and metastasis. After cocultivation, there were 26 differentially expressed genes between cancerous and hyperplastic fibroblasts. There were only three differentially expressed genes between PC‐3 cells that had been cocultivated with cancerous fibroblasts and PC‐3 cells that had been cocultivated with hyperplastic fibroblasts. The fibroblasts from cancer areas showed a different expression pattern from the characteristics reported as reactive stroma in previous studies. We found tenascin C to be downregulated, which is contrary to previous findings. TGF‐β3 and TGF‐βR3 were also downregulated, which has been associated with disturbance of TGF‐β signaling during prostate cancer progression. Cocultivation with PC‐3 cells seems to make the cancerous and hyperplastic fibroblasts more alike each other, as the number of differentially expressed genes decreases. It is desirable to find out if the reduction in differential gene expression is attributable to that hyperplastic fibroblasts become more alike the cancerous fibroblasts or vice versa. Also, we think that the lower expression levels of c‐Jun and c‐Fos in cancerous fibroblasts without coculture may cause loss of normal fibroblast differentiation, proliferation and inflammatory response, and hence, favor the proliferation and invasion of cancer cells.

Tyrosine kinase inhibitors alter adhesivity of prostatic cancer cells to extracellular matrix components.

Tyrosine kinase inhibitors (TKIs) are thought to have potential as a new generation of anti‐cancer drugs. Since invasiveness, the main characteristic of malignant behaviour, is believed to depend on altered cell‐matrix interactions, we investigated the effect of two potent TKIs, genistein and tyrphostin AG‐1478, on the interaction of prostate cancer cells with extracellular matrix components. PC‐3 and DU‐145 cells were treated with various concentrations of genistein and tyrphostin AG‐1478. Adhesion to extracellular matrix was assayed using fluorescence‐labelled cells seeded on collagen type I, collagen type IV, fibronectin, laminin and vitronectin. The expression levels of integrin β1, α2, α3 and α5 subunits were measured using flow cytometry of cells labelled with monoclonal murine antibodies. Genistein treatment reduced the ability of both cell lines to adhere to the matrix proteins tested. This effect was more pronounced for PC‐3 cells than for DU‐145 cells. Genistein treatment decreased the expression of β1 integrins by 40% in PC‐3 cells and 22% in DU‐145. AG‐1478 treatment slightly reduced the ability of DU‐145 cells to adhere, but did not decrease PC‐3 cell adhesion. Nevertheless, expression levels were reduced for most integrins tested, except the expression of α‐5, for which no significant effect was measured. Our results point to a possible role of TKIs as suppressors of prostate carcinoma cell adhesion to extracellular matrix components, by acting as inhibitors of integrin expression.

Carcinoma Metastasis - An Approach to Models

Background: Epithelium is separated from other tissues in the body by the basal membrane. When respecting this boundary, atypical epithelial growth does not cause serious illness in most cases. Therefore, carcinoma in situ is considered to be a non-malignant condition. However, the situation is quite different if the epithelial cells do not respect the natural boundaries in the tissue, a condition that is often referred to as cancer. Uncontrolled invasive growth is indeed the main characteristic of malignancy, and metastasis is in most cases the reason why cancer patients die. Materials and methods: The purpose of this article is to highlight how carcinoma cells, nature, can be classified in three steps, in respect to the first local infiltration of malignant epithelium. The literature referred to is selected on the basis that the views advocated are not controversial, and not represent individual findings. Moreover, some considerations are based on the authors own experience in clinical and molecular basic research. Results: The main characteristics of invasive cellular behavior are modified adhesion and a transition from fixed cells to a migratory phenotype. Invasion is made possible by the degradation of extracellular components. We only know fragments of the gene and phenotypic changes that enable cancers origin of behavior, but there is evidence that chemokines play a central role in the directional spread of motile cells. However, the most common characteristic of carcinoma cells is their loss of cell polarity. Interpretation: The complexity of multicellular organisms is staggering. Artificial and highly simplified model systems are therefore cancer researcher’s most important tools. To be significant, such results must be translated and verified to the in vivo situation. In addition, generality in cancer research finding must be given greater importance, since individual results, not can form the basis for new treatment regimes. Today’s biggest challenge for researchers is therefore to be able to collate the enormous diversity of molecular biological knowledge that daily runs.

Urokinase plasminogen activator receptor (uPAR) expression is reduced by tyrosine kinase inhibitors

Previously we reported that tyrosine kinase inhibitors (TKI) produced a reduction in uPA expression in prostatic cancer cells, and that TKI‐treated cells were less invasive compared to untreated cells. Nevertheless, no change in cell migration was observed when TKI‐treated cells were supplied with external uPA, thus indicating more complex mechanisms leading to decreased cell invasion. uPAR expression was measured with an enzyme‐ linked immunosorbent assay (ELISA) in PC‐3 and DU‐145 prostate carcinoma cells treated with the two TKI genistein and AG‐1478. uPAR mRNA levels were measured with real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). uPAR immunocytochemistry was used to examine the receptor distribution in cells grown on a reconstituted basal lamina. Immunocytochemistry showed an intense uPAR immunostaining in invading cells, particularly in the leading edge membrane. Treatment with genistein and AG‐1478 led to a decreased expression of uPAR in DU‐145, but not in PC‐3. Furthermore, a reduction of uPAR mRNA was found in TKI‐treated DU‐145 cells, while PC‐3 was not affected. Our results indicate a possible role of TKI as cancer suppressors by acting as a regulator of uPAR expression.

Can Drying Be an Alternative Tissue Preservation Method in Cancer Research Biobanking

The currently available methods for conservation of biobank material are mainly based on formalin fixation or the use of different freezing techniques. For molecular biological analysis, it is common to use quick freezing and low-temperature storage of the tissue materiel. This is a very energy-intensive and expensive method that requires advanced infrastructure, including monitoring and control procedures. The purpose of this work has been to study drying as an alternative process to cryogenic storage of undried biobank material, especially for use in cancer research groups. Fast freezing has been shown to be suitable to preserve the integrity of RNAs, while traditional formalin fixation preserves proteins and thus morphology in a good way. Various fresh-harvested murine tissues, such as lung, heart, skeletal muscle, liver, and kidney, were quickly frozen in liquid nitrogen and then subsequently dried at +5°C and −10°C, respectively, in a heat pump dryer. After drying, the RNA integrity was measured. The dried material was then stored for five months at +4°C and −20°C in commercial refrigerators, with subsequent measurement of RNA integrity. Dried materials were also evaluated with light microscopy and by electron microscopy with respect to tissue and cell structure. The same pattern was found for all five murine tissues. We conclude that drying at temperatures below 0°C is most careful to preserve the RNA integrity, with approximately the same RIN score of dried and non-dried samples for all five tissues. What characterized the general pattern of stored samples is that drying leads to a preservation of RNA integrity. Moreover, architecture in tissue resembled normal sections prepared from fresh tissue. In some places in the rim of the tissue sample, the lung tissue revealed alveolar-like morphology. In the electron microscope, few organelles other than the nuclei could be identified. Drying of biological material is a promising and cost-effective method for biobanks that store tissue, compared to cryogenic storage of undried material. Degradation of RNA, measured by the RIN number, is a critical factor in storing biobank tissue. In low-temperature dried material, the RIN factor is at the same level as storage of undried material at cryogenic temperatures, which is the common way of storing biobank material today. In this study, a heat pump dryer was used successfully to establish drying temperatures below and above the freezing point of the material. Further work has to be done in order to study different drying methods, drying conditions, and drying costs.

Inhibitors of Tyrosine Kinases (TKI) and Small Interfering RNAs (siRNA) are Promising Targeted Cancer Treatments

Background: In recent years, many investigators have focused on the development of drugs with the potential to influence the malignant phenotype. Clearly, our understanding of molecular and cellular processes behind such mechanisms has increased substantially through the past decades. This knowledge has led to an extensive search for targeted carcinoma treatment.Methods: This review is based on a qualified selection of articles published in internationally recognized journals. Some considerations are also based on the authors’ own experience in clinical and molecular-targeted basic research.Results: Inhibitors of tyrosine kinases (TKIs) and selective gene expressional regulators, small interfering RNAs, give hope for advances in the treatment of various carcinomas. However, it seems clear that the main challenge also in such therapy is the delivery, toxicity, uptake, and stability of small targeted molecules. These must be dealt with in order to unleash the potential of individual therapy.Conclusion: Development of TKIs and siRNAs, with effects better than or at least similar to conventional therapy must be given high priority. Tolerable side effects are also critical to successful implementation. Only through such efforts may such treatment be fully developed and find its place in the treatment of malignant tumours, solely, as primary choice, or in combination with conventional cancer therapy.

Increased levels of serum miR-148a-3p are associated with prostate cancer

Prostate cancer (PCa) is one of the most common types of cancer and the fifth leading cause of death among men worldwide. The tools for diagnosing PCa have limited value, and to improve correct diagnosis there is a need for markers that can contribute to a more precise diagnosis, which would lead to proper treatment of only those patients who need it. Micro RNA (miRNA) plays a key role in the development of cancer and is therefore a potential marker for PCa. Next‐generation sequencing was used to discover differences in miRNA expression between serum samples from PCa patients and healthy controls, and the results were validated by quantitative real‐time polymerase chain reaction. Detection of the miRNA of interest was attempted in prostate tissue by in situ hybridization. All samples were collected in collaboration with Biobank1®. By miRNA sequencing of serum samples, significant expression of some miRNAs in patients with PCa and healthy controls was detected. This study showed that miR‐148a‐3p is upregulated in men with PCa, and the miRNA is differentially expressed in PCa patients compared to healthy controls. The results also showed that miR‐148a‐3p is located in prostate tissue.