Jussi Koivunen

Protein Kinase C α/β Inhibitor Go6976 Promotes Formation of Cell Junctions and Inhibits Invasion of Urinary Bladder Carcinoma Cells

Changes in activation balance of different protein kinase C (PKC) isoenzymes have been linked to cancer development. The current study investigated the effect of different PKC inhibitors on cellular contacts in cultured high-grade urinary bladder carcinoma cells (5637 and T24). Exposure of the cells to isoenzyme-specific PKC inhibitors yielded variable results: Go6976, an inhibitor of PKCα and PKCβ isoenzymes, induced rapid clustering of cultured carcinoma cells and formation of an increased number of desmosomes and adherens junctions. Safingol, a PKCα inhibitor, had similar but less pronounced effects. In contrast, a PKCδ inhibitor, rottlerin, had an opposite effect on cell clustering and caused dissociation of cell junctions. A broad-spectrum PKC inhibitor bisindolylmaleimide I did not have any apparent effect on the morphology of the cultures or on the number of cell junctions. Additional studies with Go6976 demonstrated that inhibition of PKCα and β isoenzymes induced translocation of β1-integrin from the cell-matrix junctions and that β4-integrin was translocated to face the culture substratum. Go6976 was also highly effective in inhibiting migration of carcinoma cells and inhibited invasion through artificial basement membrane. Our results on urinary bladder carcinoma cells emphasize that Go6976 is a potential anticancer drug due to its effects on cell-cell and cell-matrix junctions, migration, and invasion. Furthermore, the results may be explained by changes in PKC activation balance promoted by inhibition of PKCα/β.

NF1 tumor suppressor protein and mRNA in skeletal tissues of developing and adult normal mouse and NF1-deficient embryos

NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1‐deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease.

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.

Congenital pseudarthrosis of neurofibromatosis type 1: Impaired osteoblast differentiation and function and altered NF1 gene expression

Three patients with neurofibromatosis 1 (NF1) were operated for congenital pseudarthrosis (PA) of the tibia. Three non-NF1 patients served as reference. Both NF1 mRNA and protein were detected in the PAs and in rows of osteoblasts and numerous osteoclasts next to the NF1-related PA arguing against inactivation of both NF1 alleles in the resident cells. Analyses on mesenchymal stem cells (MSCs) cultured from the red bone marrow of 1) next to PA of the affected NF1 tibiae, 2) the non-affected NF1 iliac crest of the same patients, and from 3) non-NF1 bone marrow demonstrated that the potential to form bone in vitro was the lowest in cells from the affected NF1-tibiae. The latter cells also displayed reduced levels of NF1 mRNA and protein, and upregulated phosphorylated p44/42 MAPK levels, consistent with an upregulated Ras-pathway. An exhaustive NF1 gene analysis detected constitutional mutation in each case, but no second hits or loss of heterozygosity were found. However, one patient displayed a mutation resulting in two potential active splice sites ultimately affecting exon 6. Interestingly, only one of the respective transcripts was detected in cells from the iliac crest, but two novel transcripts were detected in MSCs cultured from site next to PA. This finding may identify a novel mechanism how a single NF1 gene mutation may exert distinct effects on separate anatomical locations. The molecular pathogenesis of NF1-related PA apparently may not be entirely explained by second mutations or loss of heterozygosity of the NF1 gene.

Heterogeneity of Cellular Proliferation within Transitional Cell Carcinoma: Correlation of Protein Kinase C Alpha/betal Expression and Activity

A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-α and -βI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-α in 13 out of 18 samples and -βI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-α and/or -βI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-α was located in the cytoplasm, whereas PKC-βI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-α and -βI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

Altered Calcium-Mediated Cell Signaling in Keratinocytes Cultured from Patients with Neurofibromatosis Type 1

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes cultured from control persons and patients with type 1 neurofibromatosis. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in Ca(2+) signaling between these cells. Keratinocytes cultured from patients with type 1 neurofibromatosis (NF1) had a tendency to form cultures with markedly altered calcium-related signaling characteristics. Specifically, the resting Ca(2+) levels, intracellular Ca(2+) stores, capacitative calcium influx, and gap-junctional signal transduction were defective in NF1 keratinocytes. Western transfer analysis revealed apparently equal connexin 43 protein levels in normal control and in NF1 keratinocytes. Indirect immunofluorescence, however, demonstrated that connexin 43 was relatively evenly distributed in NF1 cells and did not form typical gap-junctional plaques between keratinocytes. Furthermore, the speed of the calcium wave was reduced in NF1 cells compared to normal keratinocytes. The results demonstrate that keratinocytes cultured from patients with NF1 display altered calcium-mediated signaling between cells.

NF1 Tumor Suppressor mRNA Is Targeted to the Cell-Cell Contact Zone in Ca 2 -Induced Keratinocyte Differentiation

We have previously shown that NF1 (type 1 neurofibromatosis) p21ras GTPase-activating tumor suppressor protein undergoes major relocalization during the formation of cell-cell junctions in differentiating keratinocytes in vitro. This prompted us to study the distribution of NF1 mRNA under the same conditions by in situ hybridization. In differentiating keratinocytes, the NF1 mRNA signal intensified within the cell cytoplasm within the first 0.5 to 2 hours after induction of cellular differentiation. First, the hybridization signal was evenly distributed throughout the cytoplasm. Subsequently, NF1 mRNA was gradually polarized to the cellular periphery at the side of cell-cell junctions and finally disappeared. Reappearance of NF1 mRNA was found in migrating keratinocytes forming a bilayered culture. Disruption of microfibrillar cytoskeleton, but not microtubules, caused a marked change in the subcellular distribution of NF1 mRNA. This data may suggest that intact actin microfilaments are essential for transport of NF1 mRNA to the cell periphery. This is the first study demonstrating that NF1, or any tumor suppressor mRNA, belongs to a rare group of mRNAs not targeted to free polysomes or ribosomes of the rough endoplasmic reticulum. This finding recognizes a potential way for post-transcriptional modification of NF1 expression.

Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis.

BackgroundNF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes.MethodsThe expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization.ResultsThe expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins.ConclusionsThese results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes.

Prognostic and predictive role of spatially positioned tumour infiltrating lymphocytes in metastatic HER2 positive breast cancer treated with trastuzumab

Disease outcomes of HER2+ breast cancers have dramatically changed after targeted therapies, such as trastuzumab, came to clinical practice but predictive factors for trastuzumab sensitivity and resistance are frequently unknown. Current work included metastatic breast cancer patients (n = 48), who were treated with trastuzumab and had pre-treatment tumour samples available. The tumours were immunostained for T-cell (CD3, CD8), natural killer (NK)-cell (CD56) and macrophage (CD68) markers and quantitative analysis of the immune cells was carried out using a computer-assisted image analysis in different tumour locations. High number of CD3 and CD8 positive T-cells was associated with significant survival benefit in the center of the tumour (CT) (p = 0.007, p = 0.001) but not in the invasive margin. The number of NK-cells and macrophages in the CT showed non-significant tendency towards improved survival. In subgroup analyses, high density of CD8 CT cells was associated with significant survival benefit in non-bone only disease, in TX or T1-3, and in ER+ tumours (p = 0.006, p = 0.003, p = 0.001). Moreover, high CD8 CT cell density associated significantly with long trastuzumab interruption periods in response. The results suggest important prognostic and predictive role of tumour infiltrating lymphocytes in center of the tumours in metastatic HER2+ breast cancer.

Abstract 1791: Spatially positioned tumor infiltrating lymphocytes predict survival in metastaticHER2positive breast cancer treated with trastuzumab

Background: Disease outcomes of HER2+ breast cancers have dramatically changed after targeted therapies, such as trastuzumab, came to clinical practice but predictive factors for trastuzumab sensitivity and resistance are frequently unknown. Methods: Metastatic breast cancer patients (n=48), who were treated with trastuzumab and had pre-treatment tumor samples available, were studied. The tumors were immunostained for T-cell (CD3, CD8) and NK-cell (CD56) markers and quantitative analysis of the immune cells was carried out using a computer-assisted image analysis in different tumor locations. Results: High number of CD3 and CD8 positive T-cells was associated with significant survival benefit in the center of the tumor (CT) (p=0.007, p=0.001), but not in the invasive region. NK-cell tumor infiltration was infrequent and they could not be reliably analyzed. In a subgroup analyses, high density of CD8 CT cells was associated with significant survival benefit in non-bone only disease, in TX-3, and in ER+ tumors (p=0.006, p=0.003, p=0.001). Moreover, high CD8 CT cell density was associated with good trastuzumab responses (p=0.042). Conclusion: High number of CD3 and CD8 positive tumor infiltrating lymphocytes in the CT area is associated with survival benefit in some patient groups with HER2+ breast cancer treated with trastuzumab. Furthermore, high number of CD8 CT cells predicts benefit from trastuzumab. Citation Format: Jussi Pekka Koivunen, Tiia Honkanen, Tiina Moilanen, Peeter Karihtala, Juha Vayrynen, Markus Makinen. Spatially positioned tumor infiltrating lymphocytes predict survival in metastatic HER2 positive breast cancer treated with trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2017-1791