Background and Aims: The fitness and viability of a tumor ecosystem are influenced by the spatial organization of its cells. We aimed to study the structure, architecture, and cell-cell dynamics of the heterogeneous liver cancer tumor microenvironment using spatially resolved multiplexed imaging. Approach and Results: We performed co-detection by indexing multiplexed immunofluorescence imaging on 68 HCC biopsies from Thai patients [(Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC)] as a discovery cohort, and then validated the results in an additional 190 HCC biopsies from Chinese patients [Liver Cancer Institute (LCI)]. We segmented and annotated 117,270 and 465,632 cells from the TIGER-LC and LCI cohorts, respectively. We observed 4 patient groups of TIGER-LC (IC1, IC2, IC3, and IC4) with distinct tumor-immune cellular interaction patterns. In addition, patients from IC2 and IC4 had much better overall survival than those from IC1 and IC3. Noticeably, tumor and CD8 + T-cell interactions were strongly enriched in IC2, the group with the best patient outcomes. The close proximity between the tumor and CD8 + T cells was a strong predictor of patient outcome in both the TIGER-LC and the LCI cohorts. Bulk transcriptomic data from 51 of the 68 HCC cases were used to determine tumor-specific gene expression features of our classified subtypes. Moreover, we observed that the presence of immune spatial neighborhoods in HCC as a measure of overall immune infiltration is linked to better patient prognosis. Conclusions: Highly multiplexed imaging analysis of liver cancer reveals tumor-immune cellular heterogeneity within spatial contexts, such as tumor and CD8 + T-cell interactions, which may predict patient survival.
Abstract RasGRP3, a RasGEF (Ras guanine nucleotide exchange factor), is an activator of H-Ras, R-ras and Rap1, Here, we report that its expression level was increased in multiple human melanoma cell lines, reaching a level of protein expression approaching that of Ramos cells in the case of the SK-MEL-5 melanoma line. Likewise, RasGRP3 expression was variably elevated in human melanoma tissue samples. The importance of RasGRP3 expression for the melanoma cell lines was demonstrated by down regulating its expression, which inhibited cell proliferation and blocked both colony formation in soft agar and xenograft tumor formation in immunodeficient mice in the case of both the M14 and SK-Mel-5 cell lines. Finally, RasGRP3 was involved in downstream signaling in these cells. Suppression of RasGRP3 expression reduced both basal and HGF induced AKT phosphorylation in the M14 and SK-Mel-5 cells. Consistent with these results, we found that overexpression of RasGRP3 in human primary melanocytes changed their morphology, enhanced cell proliferation, and caused xenograft tumor formation. Suppression of the RasGRP3 overexpression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. We conclude that RasGRP3 represents a potential therapeutic target in melanoma. Even more generally, the high level of expression and functional role of RasGRP3 in melanoma emphasizes that this RasGEF may play a more general role in regulation of Ras family members than had been initially appreciated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5029.
Ingenol 3-angelate (I3A) is one of the active ingredients in Euphorbia peplus, which has been used in traditional medicine. Here, we report the initial characterization of I3A as a protein kinase C (PKC) ligand. I3A bound to PKC-alpha in the presence of phosphatidylserine with high affinity; however, under these assay conditions, little PKC isoform selectivity was observed. PKC isoforms did show different sensitivity and selectivity for down-regulation by I3A and phorbol 12-myristate 13-acetate (PMA) in WEHI-231, HOP-92, and Colo-205 cells. In all of the three cell types, I3A inhibited cell proliferation with somewhat lower potency than did PMA. In intact CHO-K1 cells, I3A was able to translocate different green fluorescent protein-tagged PKC isoforms, visualized by confocal microscopy, with equal or higher potency than PMA. PKC-delta in particular showed a different pattern of translocation in response to I3A and PMA. I3A induced a higher level of secretion of the inflammatory cytokine interleukin 6 compared with PMA in the WEHI-231 cells and displayed a marked biphasic dose-response curve for the induction. I3A was unable to cause the same extent of association of the C1b domain of PKC-delta with lipids, compared with PMA or the physiological regulator diacylglycerol, and was able to partially block the association induced by these agents, measured by surface plasmon resonance. The in vitro kinase activity of PKC-alpha induced by I3A was lower than that induced by PMA. The novel pattern of behavior of I3A makes it of great interest for further evaluation.
Abstract C1 domains are the recognition motif for the second messenger diacylglycerol and for the phorbol esters. They play a critical role in the regulation of multiple families of signaling proteins such as protein kinase C, RasGRP, and the chimaerins. Atypical C1 domains preserve substantial homology with the diacylglycerol / phorbol ester responsive C1 domains but fail to bind these ligands. A recent advance in the field has been the recognition that the atypical C1 domains represent a continuum of structures ranging from those with measurable but weak phorbol ester binding affinity through those that preserve the phorbol ester binding cleft but have residues interfering with the insertion of the C1 domain into membranes and finally those that lack appropriate binding cleft geometry. STAC2 (SH3 and cysteine-rich domain 2) protein attracted our interest because, while its C1 domain has been classified as atypical, it had actually not been tested for phorbol ester binding activity and possesses marked homology to C1 domains that bind phorbol esters. The strategy for analysis has been to probe the influence of individual residues in the STAC2 C1 domain that are either not present or are unusual in the numerous phorbol ester responsive C1 domains. Reciprocal approaches have been to start either with the STAC2 C1 domain, mutate the individual residues, and look for gain of phorbol ester binding activity or to start with the PKC delta C1b domain and look for residues that cause reduction in binding affinity. The former approach is efficient if a single residue is responsible for the lack of binding activity; the latter approach is preferable if multiple residues are responsible. First, we confirmed that indeed the C1 domain of STAC2 is atypical and fails to bind phorbol ester. Second, we identified the residues in positions 11, 13, 24 25, 41 and 45 of the C1 domain of STAC2 as potential candidates for interfering with binding activity. However, replacement of none of these residues with the corresponding residues from PKC delta C1b sufficed to generate phorbol ester binding activity. One the other hand, introducing mutations into the C1b domain of PCK delta identified L24N, that caused a significant (~20-fold) decrease in the binding affinity. The probable basis for the negative effect of L24N on binding is that Leucine 24, together with Phenylalanine 13, Leucine 20 and Tryptophan 22, are all exposed at the top of the binding cleft and insert into the lipid membrane during ligand binding. The replacement of a hydrophobic residue at position 24 with a hydrophilic residue should interfere with this binding. STAC2 may thus fall into the category of proteins with atypical C1 domains that could be targeted with phorbol derivatives that do not stabilize membrane insertion. Our findings strengthen our insight into the structural basis and understanding of the interplay between the C1 domain, ligand and the membrane as a ternary complex. Citation Format: Adelle Abramovitz, Noemi Kedei, Peter M Blumberg. Understanding the structural requirements of C1 domains for phorbol ester binding, analysis of the atypical C1 domain of STAC2 [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 5344. doi:10.1158/1538-7445.AM2017-5344
31 Background: The human prostate cell line LNCaP and the human myelocytic leukemia cell line U937 differ dramatically in their responses to the two protein kinase C (PKC) targeted ligands phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 and show complex differences in the patterns of transcriptional responses that they induce. Quantitation of relative abundance of individual PKC isoforms in the two cell lines may help to link the downstream effects of the two compounds to these isoforms. Methods: Simple Western is a capillary-based automated Western system recently developed by ProteinSimple. All steps following sample preparation are fully automated in the Simple Western system, including sample loading, size-based protein separation, immunoprobing, washing, detection and data analysis. Simple Western is gel-free and blot-free, uses less amount of samples, and produces highly quantitative, reproducible information that cannot be generated using regular Western assays. Using the Simple Western system, we developed a method for absolute quantitation of endogenous proteins in cell lysates and quantified PKC isoforms in LNCaP and U937 cells. Results: PKC isoforms were measured at levels of picogram or sub-picogram per nanogram cell lysate. PKC delta was identified as the dominant PKC isoforms in both cell lines. In LNCaP cells, PKC delta expression is ~20-fold higher than PKC alpha, ~40-fold higher than PKC epsilon, and at least 20-fold higher than PKC beta. In U937 cells, PKC delta expression is similar to PKC beta, at least 200 fold higher than PKC alpha, and ~50-fold higher than PKC epsilon. Conclusions: The Simple Western system, with its high-quality data quantitation and excellent assay reproducibility, allowed us to detect both the relative abundance of the PKC isoforms and their absolute quantitation in the tested cells. It circumvents the problem that antibodies of different affinities for different proteins yield a misleading impression of relative abundance and it provides an approach to accurately correlate protein quantities with their function.
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