Christer Larsson

PKCα expression is a marker for breast cancer aggressiveness

BackgroundProtein kinase C (PKC) isoforms are potential targets for breast cancer therapy. This study was designed to evaluate which PKC isoforms might be optimal targets for different breast cancer subtypes.ResultsIn two cohorts of primary breast cancers, PKCα levels correlated to estrogen and progesterone receptor negativity, tumor grade, and proliferative activity, whereas PKCδ and PKCε did not correlate to clinicopathological parameters. Patients with PKCα-positive tumors showed poorer survival than patients with PKCα-negative tumors independently of other factors. Cell line studies demonstrated that PKCα levels are high in MDA-MB-231 and absent in T47D cells which proliferated slower than other cell lines. Furthermore, PKCα silencing reduced proliferation of MDA-MB-231 cells. PKCα inhibition or downregulation also reduced cell migration in vitro.ConclusionsPKCα is a marker for poor prognosis of breast cancer and correlates to and is important for cell functions associated with breast cancer progression.

B cell activation by outer membrane vesicles--a novel virulence mechanism.

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19+ IgD+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

Novel and classical protein kinase C isoforms have different functions in proliferation, survival and differentiation of neuroblastoma cells

To elucidate the possibility of utilizing protein kinase C (PKC) isoforms as target genes in neuroblastoma therapy, 5 neuroblastoma cell lines and neuroblastoma tumor specimens were examined for PKC isoform expression pattern and the cell lines were analyzed for sensitivity to PKC inhibition. All cell lines [IMR‐32, LAN‐2, LAN‐5, SH‐SY5Y and SK‐N‐BE(2)] expressed α, βII, δ and ϵ isoforms of PKC, while no PKCη or θ protein was detected in any cell line. PKCγ was found only in LAN‐2 cells. PKCα, βII and δ were detected in 5 neuroblastoma tumors and PKCϵ in 4 out of 5 tumors. Exposure to the PKC inhibitors GF109203X, Gö 6976 or Gö 6983 caused a decrease whereas activation of PKC with 12‐O‐tetradecanoyl phorbol 13‐acetate caused an increase in the number of neuroblastoma cells. The effect of Gö 6976 was due to both inhibited proliferation and to increased apoptosis. While GF109203X suppressed neurite outgrowth induced by a growth factor combination, Gö 6976 potentiated neurite outgrowth. Our data suggest a role for classical PKC isoforms in neuroblastoma growth and survival and for novel isoforms in neurite outgrowth. Int. J. Cancer 81:494–501, 1999.

Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) ϵ induces ADAM12 translocation to the cell surface and that catalytic activity of PKCϵ is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 μm phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCϵ could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCϵ expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCϵ mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCϵ both contain a binding site for ADAM12. These studies show that PKCϵ plays a critical role in the regulation of ADAM12 cell-surface expression.

Distinct roles of PKC isoforms in NCAM-mediated neurite outgrowth

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)‐mediated neurite outgrowth was tested using a co‐culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12‐E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM‐stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM‐mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase‐deficient forms of PKCα, βI and βII blocked NCAM‐mediated neurite extension, but had no effect on nerve growth factor (NGF)‐mediated neurite outgrowth. Expression of two PKCɛ constructs: (i) a fragment from PKCɛ encompassing the pseudosubstrate, the C1a domain (including the actin‐binding site, ABS), and parts of the V3 region, or (ii) the PKCɛ‐specific ABS blocked NCAM‐mediated neurite extension in both cases. These two constructs also partially inhibited NGF‐stimulated neuritogenesis indicating that PKCɛ is a positive regulator of both NCAM‐ and NGF‐mediated differentiation. We suggest that PKCɛ is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM‐stimulated neurite outgrowth.

The catalytic domain limits the translocation of protein kinase C alpha in response to increases in Ca2+ and diacylglycerol.

Translocation of protein kinase C (PKC) alpha, beta II, delta and epsilon fused to enhanced green fluorescent protein (EGFP) was studied in living neuroblastoma cells by confocal microscopy. Exposure to carbachol elicited transient translocation of PKC alpha-EGFP and beta II-EGFP in most of the cells, PKC delta-EGFP in a few cells and induced sustained translocation of PKC epsilon-EGFP. To monitor levels of Ca(2+) and diacylglycerol and the translocation of PKC in the same cell, the Ca(2+)-sensitive C2 domain, diacylglycerol-sensitive C1 domains and full-length PKC were fused to red, cyan and yellow fluorescent proteins respectively. PKC alpha was translocated a few seconds after the C2 domain, which represents an increase in Ca(2+). This delay was insensitive to removal of the pseudosubstrate in PKC alpha, but the isolated regulatory domain translocated simultaneously with the C2 domain. Translocation of PKC epsilon coincided with the increase in diacylglycerol. Ionomycin induced translocation of PKC alpha and the C2 domain, whereas 1,2-dioctanoylglycerol caused translocation of the C1 domains and PKC epsilon, but not PKC alpha. Experiments with individual C1 domains showed that treatment with carbachol or phorbol 12,13-dibutyrate elicited translocation of PKC alpha C1a, PKC epsilon C1a and PKC epsilon C1b, whereas PKC alpha C1b was largely insensitive to these agents. In contrast with full-length PKC alpha, the regulatory domain of PKC alpha and pseudosubstrate-devoid PKC alpha responded to the carbachol-stimulated increase in diacylglycerol.

Expression of miR-34c induces G2/M cell cycle arrest in breast cancer cells

BackgroundMicroRNA-34 is a family of three miRNAs that have been reported to function as tumor suppressor miRNAs and show decreased expression in various cancers. Here, we examine functions of miR-34c in basal-like breast cancer cells.MethodsData from The Cancer Genome Atlas (TCGA) were used for evaluation of expression in primary breast cancers. Cellular processes affected by miR-34c were investigated by thymidine incorporation, Annexin V-assays and cell cycle analysis using breast cancer cell lines. Effects on potential targets were analyzed with qPCR and Western blot.ResultsTCGA data revealed that miR-34c was expressed at lower levels in basal-like breast cancer tumors and low expression was associated with poor prognosis. Ectopic expression of miR-34c in basal-like breast cancer cell lines resulted in suppressed proliferation and increased cell death. Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase. We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines. In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest. However, protein levels of PRKCA, a predicted miR-34c target and a known regulator of breast cancer cell proliferation were not influenced by miR-34c.ConclusionsTogether, our results support the role of miR-34c as a tumor suppressor miRNA also in breast cancer.

Prognostic stromal gene signatures in breast cancer

IntroductionGlobal gene expression analysis of tumor samples has been a valuable tool to subgroup tumors and has the potential to be of prognostic and predictive value. However, tumors are heterogeneous, and homogenates will consist of several different cell types. This study was designed to obtain more refined expression data representing different compartments of the tumor.MethodsFormalin-fixed paraffin-embedded stroma-rich triple-negative breast cancer tumors were laser-microdissected, and RNA was extracted and processed to enable microarray hybridization. Genes enriched in stroma were identified and used to generate signatures by identifying correlating genes in publicly available data sets. The prognostic implications of the signature were analyzed.ResultsComparison of the expression pattern from stromal and cancer cell compartments from three tumors revealed a number of genes that were essentially specifically expressed in the respective compartments. The stroma-specific genes indicated contribution from fibroblasts, endothelial cells, and immune/inflammatory cells. The gene set was expanded by identifying correlating mRNAs using breast cancer mRNA expression data from The Cancer Genome Atlas. By iterative analyses, 16 gene signatures of highly correlating genes were characterized. Based on the gene composition, they seem to represent different cell types. In multivariate Cox proportional hazard models, two immune/inflammatory signatures had opposing hazard ratios for breast cancer recurrence also after adjusting for clinicopathological variables and molecular subgroup. The signature associated with poor prognosis consisted mainly of C1Q genes and the one associated with good prognosis contained HLA genes. This association with prognosis was seen for other cancers as well as in other breast cancer data sets.ConclusionsOur data indicate that the molecular composition of the immune response in a tumor may be a powerful predictor of cancer prognosis.

A PKCβ isoform mediates phorbol ester-induced activation of Erk1/2 and expression of neuronal differentiation genes in neuroblastoma cells

Protein kinase C (PKC) activation induces neuronal differentiation of SH‐SY5Y neuroblastoma cells. This study examines the role of PKCβ isoforms in this process. The PKCβ‐specific inhibitor LY379196 had no effect on 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐induced neurite outgrowth from SH‐SY5Y neuroblastoma cells. On the other hand, PKCβ inhibition suppressed the TPA‐stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA‐induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCβ isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells.

The Sweden Cancerome Analysis Network - Breast (SCAN-B) Initiative: a large-scale multicenter infrastructure towards implementation of breast cancer genomic analyses in the clinical routine

BackgroundBreast cancer exhibits significant molecular, pathological, and clinical heterogeneity. Current clinicopathological evaluation is imperfect for predicting outcome, which results in overtreatment for many patients, and for others, leads to death from recurrent disease. Therefore, additional criteria are needed to better personalize care and maximize treatment effectiveness and survival.MethodsTo address these challenges, the Sweden Cancerome Analysis Network - Breast (SCAN-B) consortium was initiated in 2010 as a multicenter prospective study with longsighted aims to analyze breast cancers with next-generation genomic technologies for translational research in a population-based manner and integrated with healthcare; decipher fundamental tumor biology from these analyses; utilize genomic data to develop and validate new clinically-actionable biomarker assays; and establish real-time clinical implementation of molecular diagnostic, prognostic, and predictive tests. In the first phase, we focus on molecular profiling by next-generation RNA-sequencing on the Illumina platform.ResultsIn the first 3xa0years from 30 August 2010 through 31 August 2013, we have consented and enrolled 3,979 patients with primary breast cancer at the seven hospital sites in South Sweden, representing approximately 85% of eligible patients in the catchment area. Preoperative blood samples have been collected for 3,942 (99%) patients and primary tumor specimens collected for 2,929 (74%) patients. Herein we describe the study infrastructure and protocols and present initial proof of concept results from prospective RNA sequencing including tumor molecular subtyping and detection of driver gene mutations. Prospective patient enrollment is ongoing.ConclusionsWe demonstrate that large-scale population-based collection and RNA-sequencing analysis of breast cancer is feasible. The SCAN-B Initiative should significantly reduce the time to discovery, validation, and clinical implementation of novel molecular diagnostic and predictive tests. We welcome the participation of additional comprehensive cancer treatment centers.Trial registrationClinicalTrials.gov identifier NCT02306096.