Unique cellular interactions between pancreatic cancer cells and the omentum.

Pancreatic cancer is a common cause of cancer-related mortality. Omental spread is frequent and usually represents an ominous event, leading to patient death. Omental metastasis has been studied in ovarian cancer, but data on its role in pancreatic cancer are relatively scarce and the molecular biology of this process has yet to be explored. We prepared tissue explants from human omental fat, and used conditioned medium from the explants for various in vitro and in vivo experiments designed to evaluate pancreatic cancer development, growth, and survival. Mass spectrometry identified the fat secretome, and mRNA array identified specific fat-induced molecular alternations in tumor cells. Omental fat increased pancreatic cancer cellular growth, migration, invasion, and chemoresistance. We identified diverse potential molecules secreted by the omentum, which are associated with various pro-tumorigenic biological processes. Our mRNA array identified specific omental-induced molecular alternations that are associated with cancer progression and metastasis. Omental fat increased the expression of transcription factors, mRNA of extracellular matrix proteins, and adhesion molecules. In support with our in vitro data, in vivo experiments demonstrated an increased pancreatic cancer tumor growth rate of PANC-1 cells co-cultured for 24 hours with human omental fat conditioned medium. Our results provide novel data on the role of omental tissue in omental metastases of pancreatic cancer. They imply that omental fat secreted factors induce cellular reprogramming of pancreatic cancer cells, resulting in increased tumor aggressiveness. Understanding the mechanisms of omental metastases may enable us to discover new potential targets for therapy

The Conserved Carboxy Terminus of the Capsid Domain of Human Immunodeficiency Virus Type 1 Gag Protein Is Important for Virion Assembly and Release

The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an α-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process

Omental fat CM enhances pancreatic cancer cell migration and invasion.

<p>(A) Wound healing scratch assay demonstrating the effect of omental fat CM on PANC-1 and MIA-PaCa-2 cell migration; Scale bar = 200 μm. (B); Modified Boyden chamber assays depicting the effects of omental fat CM on pancreatic cancer cell migration (<i>P<</i> .001); (C) Matrigel invasion chamber demonstrating a significant increase in invasion of PANC-1 and MIA-PaCa-2 cells pre-treated with omental fat CM (<i>P</i>< .05); (D) Increased invasion of PANC-1 and MIA-PaCa-2 cells by using omental fat CM as a chemoattractant (<i>P</i> < .01). The upper panel graphs represent the average of 5 repeated experiments ± SD, and the lower panel depicts representative images (magnification, X100).</p

Omental fat CM enhances pancreatic cancer cell growth.

<p>(A) XTT assay demonstrating a significant increase (<i>P</i>< .01) in proliferation of pancreatic cancer cells after incubation with omental fat CM, n = 7; (B) Omental fat CM markedly increased pancreatic cancer cell colony formation capacity (<i>P</i>< .01). The upper panel graphs represent the average of four repeated independent experiments ±SD, and the lower panel depicts representative images of cell colonies in soft agar (magnification, X100); (C) Omental fat CM-induced S-phase population in pancreatic cancer cells; a more pronounced effect was seen in PANC-1 cells than in MIA-PaCa-2 cells (<i>P</i>< .05). Bar plots display the data of 7 independent experiments.</p

Molecular characterization of omental fat CM-treated PANC-1 cells.

<p>(A) PCA of gene expression microarray data. The PCA graphs of global gene expression data were computed using Partek GS, version 6.6; RM control samples are shown as red spheres, n = 3; CM samples are shown as yellow spheres, n = 9. (B) Affymetrix microarray hierarchical clustering performed on mRNA of PANC-1 cells treated with omental fat CM compared to RM. A colored bar indicating the standardized log2 intensities accompanies the expression profile. (C) qRT-PCR validation of the expression levels of OPN in PANC-1 cells pretreated with omental fat CM compared to RM, n = 6. Cells were analyzed by Western blot for the expression of <i>OPN</i> protein levels. α-actinin was used as a protein loading control, n = 6.</p

Cancer-related genes identified by mRNA array analysis of PANC-1 cells pre-treated with omental fat conditioned medium (CM) versus control regular medium (RM).

<p>Cancer-related genes identified by mRNA array analysis of PANC-1 cells pre-treated with omental fat conditioned medium (CM) versus control regular medium (RM).</p

Cancer-related proteins identified by LC-MS/MS analysis of human omental fat (OF) versus subcutaneous fat (SC).

<p>Cancer-related proteins identified by LC-MS/MS analysis of human omental fat (OF) versus subcutaneous fat (SC).</p