DNA methylation topology differentiates between normal and malignant in cell models, resected human tissues, and exfoliated sputum cells of lung epithelium.

Background: Global DNA hypomethylation is a prominent feature of cancer cells including lung cancer, that has not been widely explored towards cancer diagnosis. In this study we assess the comparative distribution of global DNA methylation in normal cells versus cancer cells in various specimen models. Methods: We used in situ immunofluorescence labeling of overall 5-methylcytosine (5mC) and covisualization of global DNA (gDNA) by 4',6-diamidino-2-phenylindole (DAPI), confocal microscopy and 3D image analysis to derive 5mC/DAPI colocalization patterns in human cell lines (BEAS-2B, A549, H157) and upper respiratory epithelial cells derived from various sources (i.e., sputum from healthy and cancer patients, and resected tissues from normal parenchyma and lung tumors). Results: By introducing 5mC/DAPI colocalization index as a metric we could distinguish between normal epithelial cells and aberrantly hypomethylated cancer cells. Cultured lung cancer cells (H157 and A549) had significantly lower indices compared to normal cells (BEAS-2B). Furthermore, we were able to identify such extensively hypomethylated low-index cells in tumor tissues and the matching sputum from cancer patients. In contrast, the indices of cells derived from sputum of healthy individuals had more similarity to epithelial cells of normal parenchyma and the phenotypically normal BEAS-2B cells. Conclusions: The results suggest that 5mC topology using high-resolution image cytometry shows potential for identifying hypomethylated cancerous cells in human tissues and amongst normal cells in matching sputum, which may render a valuable surrogate for biopsied tissues. This promising feature deserves further validation in more comprehensive studies.

Protein kinase C α enhances migration of breast cancer cells through FOXC2-mediated repression of p120-catenin.

BACKGROUND: Despite recent advances in the diagnosis and treatment of breast cancer, metastasis remains the main cause of death. Since migration of tumor cells is considered a prerequisite for tumor cell invasion and metastasis, a pressing goal in tumor biology has been to elucidate factors regulating their migratory activity. Protein kinase C alpha (PKCα) is a serine-threonine protein kinase implicated in cancer metastasis and associated with poor prognosis in breast cancer patients. In this study, we set out to define the signaling axis mediated by PKCα to promote breast cancer cell migration. METHODS: Oncomine™ overexpression analysis was used to probe for PRKCA (PKCα) and FOXC2 expression in mRNA datasets. The heat map of PRKCA, FOXC2, and CTNND1 were obtained from the UC Santa Cruz platform. Survival data were obtained by PROGgene and available at http://www.compbio.iupui.edu/proggene . Markers for EMT and adherens junction were assessed by Western blotting and quantitative polymerase chain reaction. Effects of PKCα and FOXC2 on migration and invasion were assessed in vitro by transwell migration and invasion assays respectively. Cellular localization of E-cadherin and p120-catenin was determined by immunofluorescent staining. Promoter activity of p120-catenin was determined by dual luciferase assay using a previously validated p120-catenin reporter construct. Interaction between FOXC2 and p120-catenin promoter was verified by chromatin immunoprecipitation assay. RESULTS: We determined that PKCα expression is necessary to maintain the migratory and invasive phenotype of both endocrine resistant and triple negative breast cancer cell lines. FOXC2 acts as a transcriptional repressor downstream of PKCα, and represses p120-catenin expression. Consequently, loss of p120-catenin leads to destabilization of E-cadherin at the adherens junction. Inhibition of either PKCα or FOXC2 is sufficient to rescue p120-catenin expression and trigger relocalization of p120-catenin and E-cadherin to the cell membrane, resulting in reduced tumor cell migration and invasion. CONCLUSIONS: Taken together, these results suggest that breast cancer metastasis may partially be controlled through PKCα/FOXC2-dependent repression of p120-catenin and highlight the potential for PKCα signal transduction networks to be targeted for the treatment of endocrine resistant and triple negative breast cancer.

Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

BACKGROUND: Key effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC). METHODS: We used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells. RESULTS: Immunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells. CONCLUSIONS: Our data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer.

CRK SH3N Domain Diminishes Cell Invasiveness of Non-Small Cell Lung Cancer.

CRK (c-Crk) as an adaptor protein is involved in several oncogenic signal transduction pathways, conveying oncogenic signals to its downstream effectors and thereby affecting multiple cellular processes including proliferation, differentiation, and migration. For example, we have observed that CRK expression and phosphorylation influence the invasiveness of non-small cell lung cancer (NSCLC) cells. To intervene in CRK signaling pathway, we examined whether CRK protein domains can be used as therapeutic tools to interrupt CRK signaling, thus influencing the biological behavior of NSCLC cells. For this purpose, Src Homology domains of CRK-I (i.e., SH2 and SH3N domains) were overexpressed in H157, Rh2, and A549 cells. CRK-SH3N domain expression induced epithelial morphology in H157 cells and enhanced epithelial morphology of A549 and Rh2 cells as compared to cells transfected with CRK-SH2 domain or empty vector. In addition, CRK-SH3N domain expression significantly decreased the motility and invasiveness of A549 and H157 cells. Furthermore, CRK-SH3N domain expression disrupted the interaction of CRK-II with DOCK180. In summary, these data provide evidence that the CRK-SH3N domain can be used to influence the malignant phenotype of NSCLC cells and also reduce the metastatic potential of these cells.

PAK1 kinase promotes cell motility and invasiveness through CRK-II serine phosphorylation in non-small cell lung cancer cells.

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.

c-Crk proto-oncogene contributes to transcriptional repression of p120-catenin in non-small cell lung cancer cells.

As a member of adherens junction, p120-catenin (p120ctn) plays a major role in cell adhesions through stabilization of E-cadherin. p120ctn is transcriptionally down-regulated in non-small cell lung cancer (NSCLC), although the molecular mechanisms underlying p120ctn repression are incompletely defined. Here we further investigated transcriptional regulation of p120ctn in NSCLC. We prepared a promoter reporter plasmid construct that contained p120ctn promoter region from position -1082 to +320 relative to transcription start site. Through serial deletion mutation analysis of the p120ctn promoter, we pinpointed cis-acting elements involved in regulation of p120ctn. We identified transcription factor SP1 as a transcriptional repressor of p120ctn that directly binds to segment (-9 to +36) of the p120ctn promoter. SP1 can receive multiple signals from several intracellular signaling pathways. Through examination of SP1 binding partners, we identified proto-oncogene c-Crk to be involved in transcriptional down-regulation of p120ctn. RNAi mediated silencing of CRK in A549, H157 and H358 cells increased p120ctn protein levels. On the other hand, over-expression of CRK-I and CRK-II in NSCLC cells down-regulated p120ctn, an effect that was abrogated by simultaneous silencing of SP1. In summary, our data provide evidence for the role of c-Crk proto-oncogene in transcriptional repression of p120ctn that further clarifies the mechanism by which this biochemical signal promotes metastasis in NSCLC.

p120-catenin is transcriptionally downregulated by FOXC2 in non-small cell lung cancer cells.

p120-catenin (p120ctn) plays a major role in cell adhesion and motility through the regulation of E-cadherin and interaction with RhoGTPase and Rac1. p120ctn is downregulated in several malignancies including non-small cell lung cancer (NSCLC). Here, we investigated transcriptional regulation of p120ctn in NSCLC. We cloned a 1,400-bp amplicon of chromosome 11 from position -1,082 to +320 relative to the transcription start site into a firefly luciferase reporter vector and prepared serial deletion constructs to pinpoint cis-acting elements involved in the regulation of p120ctn. We transfected NSCLC cell lines and immortalized normal human respiratory epithelial cells with the abovementioned constructs. We found reduced p120ctn promoter activity, protein level, and mRNA message in lung cancer cells compared with noncancerous immortalized lung epithelial cells. Serial deletion analysis of p120ctn promoter identified a region between positions +267 and +282, which mediated the transcriptional repression of p120ctn. This region harbored putative binding sites for FOXC2 and FOXL1 transcription factors. Direct binding of FOXC2 to the p120ctn promoter between positions +267 and +282 was confirmed by electromobility shift assay. RNAi-mediated silencing of FOXC2 in A549, H157, and H358 cells resulted in increasing p120ctn promoter activity as well as mRNA and protein levels. Finally, silencing FOXC2 in these NSCLC cells enhanced E-cadherin level, which was reversed by simultaneous silencing of p120ctn. In summary, our data support the notion that FOXC2 mediates the transcriptional repression of p120ctn in NSCLC.

DNA immunization against tissue-restricted antigens enhances tumor immunity after allogeneic hemopoietic stem cell transplantation.

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP --> B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.

CTLA-4 blockade in combination with xenogeneic DNA vaccines enhances T-cell responses, tumor immunity and autoimmunity to self antigens in animal and cellular model systems.

Xenogeneic DNA vaccination can elicit tumor immunity through T cell and antibody-dependent effector mechanisms. Blockade of CTLA-4 engagement with B7 expressed on APCs has been shown to enhance T cell-dependent immunity. We investigated whether CTLA-4 blockade could increase T-cell responses and tumor immunity elicited by DNA vaccines. CTLA-4 blockade enhanced B16 tumor rejection in mice immunized against the melanoma differentiation antigens tyrosinase-related protein 2 and gp100, and this effect was stronger when anti-CTLA-4 was administered with booster vaccinations. CTLA-4 blockade also increased the T-cell responses to prostate-specific membrane antigen (PSMA) when given with the second or third vaccination. Based on these pre-clinical studies, we suggest that anti-CTLA-4 should be tested with xenogeneic DNA vaccines against cancer and that special attention should be given to sequence and schedule of administration.

GM-CSF DNA induces specific patterns of cytokines and chemokines in the skin: implications for DNA vaccines.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing the proliferation, maturation, and migration of dendritic cells, and the expansion and differentiation of B and T lymphocytes. Similar biological effects have been observed with the use of GM-CSF DNA in mouse models for therapy of cancer and infectious diseases, and its use is currently being investigated in clinical trials in combination with DNA vaccines. To further understand the adjuvant mechanisms of GM-CSF DNA, we examined early events following its administration. We found measurable levels of GM-CSF protein in the skin and muscle, as well as in serum. Measurements of other cytokine and chemokine levels revealed differential expression patterns over time. The early response was characterized by high levels of inflammatory molecules, including IL-1beta, IL-6, TNFalpha, RANTES, MIP-1alpha and MCP-1, later followed by expression of precursor Th1 cytokines, IL-12 and IL-18, concomitant with IFNgamma production. Local production of GM-CSF protein also resulted in the early recruitment of polymorphonuclear cells and later recruitment of mononuclear cells, including dendritic cells. These results have implications for understanding early events in the immune response to DNA vaccines, and provide a basis for development of new approaches to cancer vaccines, including the use of cytokine genes as adjuvants.