Human leukocyte antigen (HLA) class I molecules present antigenic peptides to cytotoxic T cells, causing lysis of malignant cells. Transplantation biology studies have implicated HLA class I molecules in cell migration, but there has been little evidence presented that they influence cancer cell migration, a contributing factor in metastasis. In this study, we examined the effect of HLA-B on pancreatic cancer cell migration. HLA-B siRNA transfection increased the migration of the S2-013 pancreatic cancer cells but, in contrast, reduced migration of the PANC-1 and MIA PaCa-2 pancreatic cancer cell lines. Integrin molecules have previously been implicated in the upregulation of pancreatic cancer cell migration, and knockdown of HLA-B in S2-013 cells heightened the expression of integrin beta 1 (ITGB1), but in the PANC-1 and MIA PaCa-2 cells HLA-B knockdown diminished ITGB1 expression. A transmembrane sequence in an S2-013 HLA-B heavy chain matches a corresponding sequence in HLA-B in the BxPC-3 pancreatic cancer cell line, and knockdown of BxPC-3 HLA-B mimics the effect of S2-013 HLA-B knockdown on migration. In total, our findings indicate that HLA-B influences the expression of ITGB1 in pancreatic cancer cells, with concurrent distinctions in transmembrane sequences and effects on the migration of the cells.

Beta 2-microglobulin (β₂m) is a component of the major histocompatibility complex (MHC) class I molecule, which presents tumor antigens to T lymphocytes to trigger cancer cell destruction. Notably, β₂m has been reported as persistently expressed, rather than down regulated, in some tumor types. For renal cell and oral squamous cell carcinomas, β₂m expression has been linked to increased migratory capabilities. The migratory ability of pancreatic cancer cells contributes to their metastatic tendencies and lethal nature. Therefore, in this study, we examined the impact of β₂m on pancreatic cancer cell migration. We found that β₂m protein is amply expressed in several human pancreatic cancer cell lines (S2-013, PANC-1, and MIA PaCa-2). Reducing β₂m expression by short interfering RNA (siRNA) transfection significantly slowed the migration of the PANC-1 and S2-013 cancer cell lines, but increased the migration of the MIA PaCa-2 cell line. The amyloid precursor-like protein 2 (APLP2) has been documented as contributing to pancreatic cancer cell migration, invasiveness, and metastasis. We have previously shown that β₂m/HLA class I/peptide complexes associate with APLP2 in S2-013 cells, and in this study we also detected their association in PANC-1 cells but not MIA PaCa-2 cells. In addition, siRNA down regulation of β₂m expression diminished the expression of APLP2 in S2-013 and PANC-1 but heightened the level of APLP2 in MIA PaCa-2 cells, consistent with our migration data and co-immunoprecipitation data. Thus, our findings indicate that β₂m regulates pancreatic cancer cell migration, and furthermore suggest that APLP2 is an intermediary in this process.

Aberrantly regulated cell motility is a hallmark of cancer cells. A hybrid agent-based model has been developed to investigate the synergistic and antagonistic cell motility-impacting effects of three microenvironment variables simultaneously: chemoattraction, haptotactic permission, and biomechanical constraint or resistance. Reflecting distinct cell-specific intracellular machinery, the cancer cells are modeled as processing a variety of spatial search strategies that respond to these three influencing factors with differential weights attached to each. While responding exclusively to chemoattraction optimizes cell displacement effectiveness, incorporating permission and resistance components becomes increasingly important with greater distance to the chemoattractant source and/or after reducing the ligand's effective diffusion coefficient. Extending this to a heterogeneous population of cells shows that displacement effectiveness increases with clonal diversity as characterized by the Shannon index. However, the resulting data can be fit best to an exponential function, suggesting that there is a level of population heterogeneity beyond which its added value to the cancer system becomes minimal as directionality ceases to increase. Possible experimental extensions and potential clinical implications are discussed.

We have previously shown that the expression of a semaphorin, known as a repelling cue in axon guidance, Sema3E, correlates with the ability to form lung metastasis in murine adenocarcinoma cell models. Now, besides providing evidence for the relevance of SEMA3E to human disease by showing that SEMA3E is frequently expressed in human cancer cell lines and solid tumors from breast cancer patients, we show biological activities of Sema3E, which support the implication of Sema3E in tumor progression and metastasis. In vivo, expression of Sema3E in mammary adenocarcinoma cells induces the ability to form experimental lung metastasis, and in vitro, the Sema3E protein exhibits both migration and growth promoting activity on endothelial cells and pheochromocytoma cells. This represents the first evidence of a metastasis-promoting function of a class 3 semaphorin, as this class of genes has hitherto been implicated in tumor biology only as tumor suppressors and negative regulators of growth. Moreover, we show that the full-size Sema3E protein is converted into a p61-Sema3E isoform due to furin-dependent processing, and by analyzing processing-deficient and truncated forms, we show that the generation of p61-Sema3E is required and sufficient for the function of Sema3E in lung metastasis, cell migration, invasive growth, and extracellular signal-regulated kinase 1/2 activation of endothelial cells. These findings suggest that certain breast cancer cells may increase their lung-colonizing ability by converting the growth repellent, Sema3E, into a growth attractant and point to a type of semaphorin signaling different from the conventional signaling induced by full-size dimeric class 3 semaphorins.

Cell lines with high metastatic capacity to the lung were established by sequential passage of a human pancreatic cancer cell line (SUIT-2) through the lung of a nude mouse, via the lateral tail vein and from a subcutaneous inoculum. Cells of the parental SUIT-2 and sublines S2-VPx (x-cycle selection from SUIT-2 cells, by Vein-Pulmonary metastasis-culture) and S2-CPx (x-cycle selection, by Cutis-Pulmonary metastasis-culture) were injected intravenously or subcutaneously into nude mice to produce experimental or spontaneous lung metastasis. The S2-VP10 cell line produced pulmonary metastases in 100% of the nude mice, when injected intravenously. It failed, however, to produce more lung colonies than its parent cell line, when injected subcutaneously. The S2-CP8 cell line produced extensive pulmonary metastases in 100% of the nude mice, when injected either intravenously or subcutaneously. This study indicates that the nude mouse provided a good model for in vivo selection of metastatic cells from SUIT-2 cells both experimentally and spontaneously, and that the SUIT-2, S2-VPx, and S2-CPx cell lines will be valuable in the study of human cancer metastasis. We previously reported high levels of ezrin expression in the S2-VP10 and S2-CP8 cell lines. Here we show that these cell lines exhibit a greater capacity to invade or attach to various extracellular matrix components than the parent SUIT-2 cells. The S2-CP8 cell lines also exhibit greater level of type-I and type-IV collagen-degrading activity than the parent SUIT-2 cell line and the S2-VP10 cell line, which shows similar collagen-degrading activity to the parent SUIT-2 cells. In RT-PCR studies, SUIT-2, S2-CP8 and S2-VP10 cell lines constitutively expressed many matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP7, MMP-9, MMP-10 and MMP-14). These results suggest that some parameters that enhance adhesion and invasion are important to both experimental and spontaneous metastasis and the collagen degrading enzymes are predicted to play a key-role during spontaneous metastasis.

Pancreatic adenocarcinoma (PAC) is generally refractory to most chemotherapeutic agents, including docetaxel (Taxotere; TXT). Specific mechanisms for TXT-related drug resistance in PAC have not been defined. The hypothesis of this study was that PAC resistance to TXT is primarily related to P-glycoprotein (P-gp), the expression product of multiple drug resistance (MDR)-1, as opposed to lung resistance protein (LRP) or multidrug resistance protein (MRP).

The sensitivity of the PAC cell line SUIT-2 and its sublines to TXT, doxorubicin (DOX) and 5-fluorouracil (5-FU) was evaluated with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. MDR1 (P-gp), MRP, LRP, and beta-tubulin isotype gene expressions were detected at the messenger RNA level by reverse transcription-polymerase chain reaction (RT-PCR). Verapamil and indomethacin (IMC) were used to test the functionality of P-gp and MRP, respectively.

The SUIT-2 subline S-020 and the TXT-selected SUIT-2 cell line S2/TXT were significantly resistant to TXT. Both showed cross-resistance to DOX but no resistance to 5-FU. RT-PCR demonstrated strong expression of P-gp in S-020 and S2/TXT and weaker or no expression in other cells lines. MRP and LRP expression was found in most of these cell lines but had no relationship to the TXT resistance. TXT resistance in S2-020 and S2/TXT could be reversed by verapamil but not by IMC. Levels of beta-tubulin isotype II and III were increased in S2/TXT compared with S-020 and SUIT-2.

Intrinsic and acquired TXT resistance is primarily mediated by P-gp, but not by MRP or LRP, and is markedly reversed by the P-gp modulator verapamil. Hence future related studies should focus on the use of agents that block the transporter action of P-gp.

Previous investigations have suggested that expression of matrix metalloproteinases (MMPs) may be related to increased invasiveness of various tumors. This study evaluated a possible relation between pancreatic tumor cell invasiveness and MMPs.

A Matrigel invasion assay was performed with pancreatic tumor cell line SUIT-2 and its sublines S2-007, S2-013, S2-020, and S2-028. The degree of invasiveness of stimulated and unstimulated cell lines was correlated with MMP gene expression measured by RT-PCR and MMP protein product measured by gelatin zymography. Cell lines were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), concanavalin (Con-A), and polymerized collagen type I gel (Vitrogen).

For SUIT-2, S2-007, S2-013, S2-020, and S2-028, 3.2, 1.0, 4.1, 6.4, and 0.4%, respectively, of the cells invaded the Matrigel membrane. TPA, Con-A, and Vitrogen resulted in the up-regulation of MMP-2 in S2-020. TPA and Vitrogen resulted in up-regulation of MMP-9 in each of the cell lines, while Con-A could up-regulate MMP-9 expression only in SUIT-2. There was no constitutive expression of either MMP-2 or MMP-9 in SUIT-2 or its sublines. There was a positive relationship between Matrigel invasiveness and up-regulation of MMP-2 and MMP-9 expression.

These data suggest that, while MMP-2 and MMP-9 are not constitutively expressed in pancreatic carcinoma cell lines, they may be up-regulated by TPA, Con-A, and Vitrogen. Since MMP-2 and MMP-9 expression correlated with degree of tumor cell invasiveness, the ability to up-regulate MMP-2 and MMP-9 expression may play a role in facilitating pancreatic tumor cell invasion.

MUC1 mucin expression has been shown to be associated clinicopathologically with metastasis and poor clinical outcome in a variety of tumors. To further investigate this finding experimentally, human pancreatic cancer S2-013 cells overexpressing MUC1 were used for spontaneous metastatic potential in nude mice. It was found that the number of lung metastases of MUC1 transfectants was significantly higher than that of control cells. To analyze the molecular mechanisms that underlie the increased metastatic activity, in vitro adhesion assays were performed. MUC1 mucin expression enhancedin vitro invasiveness and motility of S2-013 cells, and decreased the binding of S2-013 cells to type I collagen, Type IV collagen and laminin. Similar effects were not observed for cells expressing tandem repeat-deleted MUC1 cDNA. Adhesion properties were abolished by benzyl-alpha-GalNAc treatment, indicating that glycosylation of the extracellular domain of MUC1 was essential for these biological adhesive functions. Our data support the hypothesis that MUC1 expression contributes to the metastatic ability of pancreatic cancer cells.

Ezrin is a membrane cytoskeleton crosslinker protein that is a member of the ERM (ezrin/radixin/moesin) family. Ezrin binds adhesion molecules such as CD43, CD44, ICAM-1, and ICAM-2, which are implicated in cell migration and metastasis. Ezrin is expressed by many tumor cell lines; however, little is known about the function of ezrin in tumorigenesis and metastasis. Here, we investigated expression of ezrin in pancreatic adenocarcinoma cell lines of different metastatic potential. Among 16 pancreatic adenocarcinoma cell lines, several cell lines showed strong expression of ezrin. Two cell lines with high metastatic potential, S2-CP9 and S2-VP10, showed very high levels of ezrin mRNA and protein, whereas other sublines showed lower levels. There was no relationship between the expression levels of ezrin and the differentiation grades of the cell lines. These results suggest that there is a relationship between high expression of ezrin and metastatic potential of pancreatic carcinomas.

Tumor cell-derived collagenase stimulatory factor (TCSF) stimulates in vitro the biosynthesis of various matrix metalloproteinases involved in tumor invasion, such as interstitial collagenase, gelatinase A, and stromelysin 1. The expression of TCSF mRNAs was studied in vivo, using in situ hybridization and Northern blotting analysis, in seven normal tissues and in 22 squamous cell carcinomas of the lung, and in seven benign proliferations and in 22 ductal carcinomas of the mammary gland. By in situ hybridization, TCSF mRNAs were detected in 40 of 44 carcinomas, in pre-invasive and invasive cancer cells of both lung and breast cancers. TCSF mRNAs and gelatinase A mRNAs were both visualized in the same areas in serial sections in breast cancers, and were expressed by different cells, tumor cells, and fibroblasts. The histological results were confirmed by Northern blot analysis, which showed a higher expression of TCSF mRNAs in cancers than in benign and normal tissues. These observations support the hypothesis that TCSF is an important factor in lung and breast tumor progression.

DNA fingerprinting can be used to detect genetic rearrangements in cancer that may be associated with activation of oncogenes and inactivation of tumour suppressor genes. We have developed a fingerprinting strategy based on polymerase chain reaction (PCR) amplification of genomic DNA with primers specific for the Alu repeat sequences, which are highly abundant in the human genome. This has been applied to DNA from pancreatic cancer and paired normal samples to isolate and identify fragments of genomic DNA rearranged in the malignant cells. These fragments have been sequenced and used as probes to isolate hybridising clones from gridded bacteriophage P1, phage artificial chromosome, and cosmid libraries for fluorescent in situ hybridisation mapping and the identification of expressed sequences. Further characterisation has identified a putative novel gene (ART1) that is up-regulated specifically in pancreatic cancer as well as another sequence with similarity to genes involved in differentiation (POU domains). In conclusion, we suggest that Alu-PCR fingerprinting may be a useful technique for the identification of genes involved in tumourigenesis.

The isoflavinoid genistein is a protein-tyrosine kinase inhibitor which has been identified as a putative cancer prevention agent. Its consumption is associated with a low incidence of clinical metastatic prostate cancer in the face of a sustained high incidence of organ-confined prostate cancer. We therefore undertook studies to examine genistein's effect upon cell adhesion as one possible mechanism by which it could be acting as an antimetastatic agent. A morphogenic analysis revealed that genistein caused cell flattening in a variety of cell lines: PC3-M, PC3, and DU-145 prostate carcinoma cells, as well as MCF-7 breast carcinoma cells. Mechanistic studies focused on the highly metastatic PC3-M cell line, and revealed that cell flattening was accompanied by an increase in cell adhesion. Further investigations demonstrated that focal adhesion kinase (FAK) accumulated in areas of focal cell attachment, and that this accumulation occurred only when cells were actively undergoing genistein-mediated morphologic change. Concurrent formation of a complex between the cell attachment molecule, beta-1-integrin, and FAK was shown to occur, and to correlate with transient activation of FAK activity. Genistein is presented as a novel investigative tool for use in the study of molecular events involved in the process of cell adhesion.