Protein kinase C inhibitor sotrastaurin selectively inhibits the growth of CD79 mutant diffuse large B-cell lymphomas.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G1-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.

SERPINB5 and AKAP12 - expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma.

BACKGROUND: Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. METHODS: Metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. RESULTS: In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05). CONCLUSIONS: AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.

DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction.

DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer.

Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential.

Adhesive and invasive characteristics appear to be crucial for organ-specific metastasis formation. Using intravital microscopy we investigated the relation between the metastatic potential of colon carcinoma cells and their adhesive and invasive behavior during early steps of metastasis within microvasculatures of rat liver, lung, intestine, skin, muscle, spleen, and kidney in vivo. Colon carcinoma cells with low (HT-29P), intermediate (KM-12C), and high (HT-29LMM, KM-12L4) metastatic potential were injected into nude or Sprague-Dawley rats. Initial interactions with host organ microvasculatures were semiquantitatively analyzed throughout 20 to 30 minutes. Circulating cells passed microvessels in all observed organs without size restriction. All cell lines showed high adhesion rates, independent from their metastatic potential, within liver and lung but very rarely in other organs. Diameters of involved microvessels were larger than diameters of adherent tumor cells. Cell extravasation of highly metastatic HT-29LMM and KM-12L4 cells into liver parenchyma was significantly higher compared to low metastatic cells (P<0.05). Our results indicate that colon carcinoma cells can arrest in target organs without size restriction. Cell adhesion of circulating tumor cells occurred in metastatic target organs only, likely attributable to specific interactions. Migration into target organs correlated with their metastatic potential.

Alphavbeta5-integrins mediate early steps of metastasis formation.

Tumour cell adhesion within the microvasculature of host organs, its stabilisation and cell invasion into the host organs, appear to be important steps in the formation of distant metastases. Intravital fluorescence-video microscopy was used to investigate the early steps in metastasis formation of colon carcinoma cells within the liver, which is the main target organ of colorectal carcinomas. The involvement of alphav-integrins was analysed in vivo using HT-29 cells after treatment with different function-blocking antibodies [pan-alphav (n=9 animals), specific alphavbeta3 (n=8 animals) and alphavbeta5 (n=8 animals)] or linear Arg-Gly-Asp (RGD)-containing peptides (RGD-peptides) (n=6 animals). Treatment with anti-alphav and anti-alphavbeta5 antibodies resulted in significantly (P<0.001) decreased tumour cell adhesion in vivo within the hepatic microvasculature. Cells treated with anti-alphavbeta3 antibodies or unspecific immunoglobulin-G (IgG) did not show significant changes in their adhesive properties. Furthermore, inhibition of cell adhesion was achieved by linear RGD-peptides in a dose-dependent manner. Relative numbers of migrated cells were not affected by any of the treatments. These results suggest that alphav-integrins, especially alphavbeta5, can influence the ability of circulating tumour cells to adhere within the hepatic microvessels. In contrast, migration of adherent cells into the liver parenchyma was not affected by alphav-integrin inhibition. Our findings support the hypothesis that specific interactions between circulating tumour cells and host organs are required for organ-specific tumour cell arrest.

Focal adhesion kinase regulates metastatic adhesion of carcinoma cells within liver sinusoids.

Organ-specific tumor cell adhesion to extracellular matrix (ECM) components and cell migration into host organs often involve integrin-mediated cellular processes that can be modified by environmental conditions acting on metastasizing tumor cells, such as shear forces within the blood circulation. Since the focal adhesion kinase (FAK) appears to be essential for the regulation of the integrin-mediated adhesive and migratory properties of tumor cells, its role in early steps of the metastatic cascade was investigated using in vitro and in vivo approaches. Human colon and hepatocellular carcinoma cells were used to study adhesive properties under static conditions and in a parallel plate laminar flow chamber in vitro. In addition, intravital fluorescence microscopy was used to investigate early interactions between circulating tumor cells and the microvasculature of potential target organs in vivo. Shear forces caused by hydrodynamic fluid flow induced Tyr-hyperphosphorylation of FAK in cell monolayers. Reduced expression of FAK or its endogenous inhibition by FAK-related non-kinase (FRNK) interfered with early adhesion events to extracellular matrix components under flow conditions. In contrast, tumor cell adhesion to endothelial cells under these conditions was not affected. Furthermore, down-regulation of FAK inhibited metastatic cell adhesion in vivo within the liver sinusoids. In summary, FAK appears to be involved in early events of integrin-mediated adhesion of circulating carcinoma cells under fluid flow in vitro and in vivo. This kinase may take part in the establishment of definitive adhesive interactions that enable adherent tumor cells to resist fluid shear forces, resulting in an organ-specific formation of distant metastases.

Integrins can directly mediate metastatic tumor cell adhesion within the liver sinusoids.

Tumor cells can show different malignant properties regarding their ability for organ-specific metastasis formation. Their adhesive and invasive characteristics mediated by various cell adhesion molecules appear to be crucial for this process. Using intravital fluorescence microscopy, we analyzed the adhesive and invasive interactions of circulating human colon carcinoma cells within the microvasculature of the liver in rats. The involvement of different cell adhesion molecules in specific tumor cell-host organ interactions was investigated. Single-cell suspensions of human colon carcinoma with low (HT-29P) and high (HT-29LMM) metastatic potential were fluorescence labeled with calcein-AM and intra-arterially injected into Sprague-Dawley rats. Initial interactions between different cell lines and the microvasculature of the liver were observed over 30 minutes and semiquantitatively analyzed. Different integrin subunits, carbohydrate ligands, and vascular cell adhesion molecule-1 were inhibited using function-blocking antibodies or by enzymatic removal. Inhibition of sialyl-Lewis(a) (sLe(a)) or enzymatic removal of selectin carbohydrate ligands significantly reduced metastatic cell adhesion. In addition, alpha6-, beta1-, and beta4-integrins can directly mediate cell adhesion within the hepatic microcirculation. Furthermore, alpha2-, alpha6-, beta1-, and beta4-integrins are involved in early tumor cell extravasation into the liver parenchyma. Organ-specific formation of colorectal metastases appears to be mainly mediated by specific interactions between circulating carcinoma cells and the vessel wall of target organs but not mechanical entrapment. Selectin-sLe(a) interactions with sinusoidal endothelial cells can play a key role in organ-specific targeting, but direct integrin-mediated cell adhesion to extracellular matrix components in the space of Disse appears to be required for the successful formation of liver metastases.

Integrity of actin fibers and microtubules influences metastatic tumor cell adhesion.

Tumor cell adhesion within host organ microvasculature, its stabilization and invasion into host organ parenchyma appear to be important steps during formation of distant metastasis. These interactions of circulating tumor cells with the host organs occur in the presence of fluid shear forces and soluble and cellular environmental conditions of the blood that can modulate their cellular responses and possibly their metastatic efficiency. Cytoskeletal components, such as actin filaments and microtubules, can regulate biophysical characteristics and cellular signaling of the circulating cells. Therefore, we investigated the role of these cytoskeletal structures for early steps during metastasis formation in vivo and in vitro. Using an intravital observation technique, tumor cell adhesion of colon carcinoma cells within the hepatic microcirculation of rats and their invasion into liver parenchyma was observed. Disruption of actin filaments increased cell adhesion, whereas tubulin disruption inhibited adhesive interactions in vivo. The impairment of the cytoskeleton modulated adhesion-mediated cell signaling via focal adhesion kinase (FAK) and paxillin under flow conditions in vitro. In the presence of fluid flow, focal adhesions were enlarged and hyperphosphorylated, whereas stress fibers were reduced compared to static cell adhesion. Disruption of microtubules, however, partially inhibited these effects. Combining the in vivo and in vitro results, our study suggested that changes in cell rigidity and avidity of cell adhesion molecules after disruption of cytoskeletal components appear to be more important for initial adhesive interactions in vivo than their interference with adhesion-mediated cellular signal transduction.